MICRORNA-BASED METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF SOLID CANCERS

METHODES FONDEES SUR LA MICROARN ET COMPOSITION POUR LE DIAGNOSTIC ET LE TRAITEMENT DE CANCERS SOLIDES

English Abstract

The present invention provides novel methods and compositions for the
diagnosis and
treatment of solid cancers. The invention also provides methods of identifying
inhibitors of
tumorigenesis.

Claims

THE EMBODIMENTS OF THE PRESENT INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMSED ARE DEFINED AS FOLLOWS:
1. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. comparing the level of at least miR-21 and miR-17-5p gene products in a
test
sample to a control level of at least miR-21 and miR-17-5p gene products;
and
b. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
other than stomach cancer if the level of at least miR-21 and miR-17-5p gene
products in the test sample from the subject is greater than the level of miR-
21 and miR-17-5p gene products of the control.
2. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. reverse transcribing at least miR-21 and miR-17-5p from a test sample
obtained from the subject to provide at least miR-21 and miR-17-5p target
oligonucleotides;
b. hybridizing at least the miR-21 and miR-17-5p target oligodeoxynucleotides
to a microarray comprising miRNA-specific probe oligonucleotides that
include at least miR-21 and miR-17-5p RNA specific probe oligonucleotides
to provide a hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; colon cancer; lung
cancer; pancreas cancer; and prostate cancer, if the level of at least miR-21
and miR-17-5p gene products in the test sample from the subject is greater
than the level of miR-21 and miR-17-5p gene products of the control.
3. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. comparing the level of at least miR-210 gene product in a test sample to
a
control level of at least miR-210 gene products; and
b. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer, if the
level of at least miR-210 gene product in the test sample from the subject is
greater than the level of miR-210 gene product of the control.
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4. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. reverse transcribing at least miR-210 from a test sample obtained from the
subject to provide at least miR-210 target oligonucleotides;
b. hybridizing at least the miR-210 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
210 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer if the
level of at least miR-210 gene product in the test sample from the subject is
greater than the level of miR-210 gene product of the control.
5. A method of diagnosing whether a subject has, or is at risk for
developing, a solid
cancer, comprising:
a. comparing the level of at least miR-213 gene product in a test sample to
a
control level of at least miR-213 gene products; and
b. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer, if the
level of at least miR-213 gene product in the test sample from the subject is
greater than the level of miR-213 gene product of the control.
6. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. reverse transcribing at least miR-213 from a test sample obtained from the
subject to provide at least miR-213 target oligonucleotides;
b. hybridizing at least the miR-213 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
213 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer, if the
level of at least miR-213 gene product in the test sample from the subject is
greater than the level of miR-213 gene product of the control.
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4. A method of diagnosing whether a subject has, or is at risk for
developing, a solid
cancer, comprising:
a. reverse transcribing at least miR-210 from a test sample obtained from the
subject to provide at least miR-210 target oligonucleotides;
b. hybridizing at least the miR-210 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
210 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer if the
level of at least miR-210 gene product in the test sample from the subject is
greater than the level of miR-210 gene product of the control.
5. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. comparing the level of at least miR-213 gene product in a test sample to
a
control level of at least miR-213 gene products; and
b. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer, if the
level of at least miR-213 gene product in the test sample from the subject is
greater than the level of miR-213 gene product of the control.
6. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. reverse transcribing at least miR-213 from a test sample obtained from the
subject to provide at least miR-213 target oligonucleotides;
b. hybridizing at least the miR-213 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
213 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer, if the
level of at least miR-213 gene product in the test sample from the subject is
greater than the level of miR-213 gene product of the control.
108

4. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. reverse transcribing at least miR-210 from a test sample obtained from the
subject to provide at least miR-210 target oligonucleotides;
b. hybridizing at least the miR-210 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
210 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer if the
level of at least miR-210 gene product in the test sample from the subject is
greater than the level of miR-210 gene product of the control.
5. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. comparing the level of at least miR-213 gene product in a test sample
to a
control level of at least miR-213 gene products; and
b. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer, if the
level of at least miR-213 gene product in the test sample from the subject is
greater than the level of miR-213 gene product of the control.
6. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. reverse transcribing at least miR-213 from a test sample obtained from the
subject to provide at least miR-213 target oligonucleotides;
b. hybridizing at least the miR-213 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
213 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: breast cancer; and lung cancer, if the
level of at least miR-213 gene product in the test sample from the subject is
greater than the level of miR-213 gene product of the control.
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7. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. comparing the level of at least miR-223 gene products in a test sample
to a
control level of at least miR-223 gene products; and
b. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
other than breast or lung cancer if the level of at least miR-223 gene
products
in the test sample from the subject is greater than the level of miR-223 gene
products of the control.
8. A method of diagnosing whether a subject has, or is at risk for developing,
a solid
cancer, comprising:
a. reverse transcribing at least miR-223 from a test sample obtained from the
subject to provide at least miR-223 target oligonucleotides;
b. hybridizing at least the miR-223 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
223 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, a solid
cancer
selected from the group consisting of: colon cancer; stomach cancer; pancreas
cancer; and prostate cancer, if the level of at least miR-223 gene products in
the test sample from the subject is greater than the level of miR-223 gene
products of the control.
9. A method of diagnosing whether a subject has, or is at risk for developing
breast
cancer, comprising:
a. comparing the level of at least miR-21 gene product in a test sample to
a
control level of at least miR-21 gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least miR-21 gene product in the test sample from the
subject
is greater than the level of miR-21 gene product of the control.
10. A method of diagnosing whether a subject has, or is at risk for developing
breast
cancer, comprising:
a. reverse transcribing at least miR-21 from a test sample obtained from the
subject to provide at least miR-21 target oligonucleotides;
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b. hybridizing at least the miR-21 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
21 RNA specific probe oligonucleotides to provide a hybridization profile for
the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least miR-21 gene products in the test sample from the
subject is greater than the level of miR-21 gene products of the control.
11. A method of diagnosing whether a subject has, or is at risk for developing
breast
cancer, comprising:
a. comparing the level of at least miR-125-1 gene product in a test
sample to a
control level of at least miR-125-1 gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least miR-125-1 gene product in the test sample from the
subject is greater than the level of miR-125-1 gene product of the control.
12. A method of diagnosing whether a subject has, or is at risk for developing
breast
cancer, comprising:
a. reverse transcribing at least miR-125-1 from a test sample obtained from
the
subject to provide at least miR-125-1 target oligonucleotides;
b. hybridizing at least the miR-125-1 target oligodeoxynucleotides to a
microarray comprising miRNA-specific probe oligonucleotides that include
at least miR-125-1 RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least miR-125-1 gene products in the test sample from the
subject is greater than the level of miR-125-1 gene products of the control.
13. A method of diagnosing whether a subject has, or is at risk for developing
breast
cancer, comprising:
a. reverse transcribing at least miR-125b-2 from a test sample obtained from
the
subject to provide at least miR-125b-2 target oligonucleotides;
b. hybridizing at least the miR-125b-2 target oligodeoxynucleotides to a
microarray comprising miRNA-specific probe oligonucleotides that include
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at least miR-125b-2 RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least miR-125b-2 gene products in the test sample from the
subject is greater than the level of miR-125b-2 gene products of the control.
14. A method of diagnosing whether a subject has, or is at risk for developing
breast
cancer, comprising:
a. reverse transcribing at least miR-145 from a test sample obtained from the
subject to provide at least miR-145 target oligonucleotides;
b. hybridizing at least the miR-145 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
145 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least miR-145 gene products in the test sample from the
subject is greater than the level of miR-145 gene products of the control.
15. A method of diagnosing whether a subject has, or is at risk for developing
pancreatic
cancer, comprising:
a. comparing
the level of at least miR-103-1 gene product in a test sample from
a subject to a control level of at least miR-103-1 gene product; and
b. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least miR-103-1 gene product in the test sample from
the subject is greater than the control level of miR-103-1 gene product.
16. A method of diagnosing whether a subject has, or is at risk for
developing, a solid
cancer, comprising:
a. reverse transcribing at least miR-103-1 from a test sample obtained from
the
subject to provide at least miR-103-1 target oligonucleotides;
b. hybridizing at least the miR-103-1 target oligodeoxynucleotides to a
microarray comprising miRNA-specific probe oligonucleotides that include
at least miR-103-1 RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
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c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least miR-103-1 gene products in the test sample
from
the subject is greater than the level of miR-103-1 gene products of the
control.
17. A method of diagnosing whether a subject has, or is at risk for developing
pancreatic
cancer, comprising:
a. comparing the level of at least miR-103-2 gene product in a test sample
to a
control level of at least miR-103-2 gene product; and
b. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least miR-103-2 gene product in the test sample from
the subject is greater than the level of miR-103-2 gene product of the
control.
18. A method of diagnosing whether a subject has, or is at risk for
developing, a solid
cancer, comprising:
a. reverse transcribing at least miR-103-2 from a test sample obtained from
the
subject to provide at least miR-103-2 target oligonucleotides;
b. hybridizing at least the miR-103-2 target oligodeoxynucleotides to a
microarray comprising miRNA-specific probe oligonucleotides that include
at least miR-103-2 RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least miR-103-2 gene products in the test sample
from
the subject is greater than the level of miR-103-2 gene products of the
control.
19. A method of diagnosing whether a subject has, or is at risk for developing
pancreatic
cancer, comprising:
a. comparing the level of at least miR-24-2 gene product in a test sample
to a
control level of at least miR-24-2 gene product; and
b. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least miR-24-2 gene product in the test sample from
the subject is greater than the level of miR-24-2 gene product of the control.
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20. A method of diagnosing whether a subject has, or is at risk for
developing, a solid
cancer, comprising:
a. reverse transcribing at least miR-24-2 from a test sample obtained from the
subject to provide at least miR-24-2 target oligonucleotides;
b. hybridizing at least the miR-24-2 target oligodeoxynucleotides to a
microarray comprising miRNA-specific probe oligonucleotides that include
at least miR-24-2 RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least miR-24-2 gene products in the test sample from
the subject is greater than the level of miR-24-2 gene products of the
control.
21. A method of diagnosing whether a subject has, or is at risk for developing
pancreatic
cancer, comprising:
a. comparing the level of at least miR-107 gene product in a test sample
to a
control level of at least miR-107 gene product; and
b. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least miR-107 gene product in the test sample from
the subject is greater than the level of miR-107 gene product of the control.
22. A method of diagnosing whether a subject has, or is at risk for
developing, a solid
cancer, comprising:
a. reverse transcribing at least miR-107 from a test sample obtained from the
subject to provide at least miR-107 target oligonucleotides;
b. hybridizing at least the miR-107 target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides that include at least miR-
107 RNA specific probe oligonucleotides to provide a hybridization profile
for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least miR-107 gene products in the test sample from
the subject is greater than the level of miR-107 gene products of the control.
23. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
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a. comparing the level of at least one miR gene product in a test sample
from a
subject to a control level of at least one corresponding miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer,
if the level of the at least one miR gene product in the test sample from the
subject is greater than a control level of the corresponding miR gene product,
wherein the miR gene product is selected from the group consisting of: miR-
21; miR-29b-2; miR-146; miR-125b-2; miR-125b-1; miR-10b; miR-145;
miR-181a; miR-140; miR-213; miR-29a prec; miR-181b-1; miR-199b; miR-
29b-1; miR-130a; miR-155; let-7a-2; miR-205; miR-29c; miR-224; miR-100;
miR-31; miR-30c; miR-17-5p; miR-210; miR-122a; and miR-16-2.
24. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. reverse transcribing at least one miR gene product from a test sample from
the subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least one miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least one miR gene product in the test sample is greater
than
the control level of one miR gene product, wherein the miR gene product is
selected from the group consisting of: miR-21; miR-29b-2; miR-146; miR-
125b-2; miR-125b-1; miR-10b; miR-145; miR-181a; miR-140; miR-213;
miR-29a prec; miR-181b-1; miR-199b; miR-29b-1; miR-130a; miR-155; let-
7a-2; miR-205; miR-29c; miR-224; miR-100; miR-31; miR-30c; miR-17-5p;
miR-210; miR-122a; and miR-16-2.
25. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. comparing the level of at least miR-29b-2 gene product in a test sample
from
a subject to a control level of at least one miR-29b-2 gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer,
if the level of at least miR-29b-2 gene product in the test sample from the
subject is greater than the control level of miR-29b-2 gene product.
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26. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. reverse transcribing at least miR-29b-2 gene product from a test sample
from
a subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least miR-29b-2 target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miR-29b-2 RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least one miR gene product in the hybridization profile
from
the test sample is greater than the control level of miR-29b-2 gene product.
27. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. comparing the level of at least one miR gene product in a test sample from
a
subject to a control level of at least one corresponding miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, colon
cancer,
if the level of the at least one miR gene product in the test sample from the
subject is greater than a control level of the corresponding miR gene product,
wherein the miR gene product is selected from the group consisting of: miR-
24-1; miR-29b-2; miR-20a; miR-10a; miR-32; miR-203; miR-106a; miR-17-
5p; miR-30c; miR-223; miR-126; miR-128b; miR-21; miR-24-2; miR-99b
prec; miR-155; miR-213; miR-150; miR-107; miR-191; miR-221; and miR-
9-3.
28. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. reverse transcribing at least one miR gene product from a test sample from
the subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least one miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
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d. diagnosing whether the subject has, or is at risk for developing, colon
cancer
if the level of at least one miR gene product in the test sample is greater
than
the control level of one miR gene product, wherein the miR gene product is
selected from the group consisting of: miR-24-1; miR-29b-2; miR-20a; miR-
10a; miR-32; miR-203; miR-106a; miR-17-5p; miR-30c; miR-223; miR-126;
miR-128b; miR-21; miR-24-2; miR-99b prec; miR-155; miR-213; miR-150;
miR-107; miR-191; miR-221; and miR-9-3.
29. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a, comparing the level of at least miR-24-1 gene product in a test sample
from a
subject to a control level of at least one miR-24-1 gene product; and
b. diagnosing whether the subject has, or is at risk for developing, colon
cancer,
if the level of at least miR-24-1 gene product in the test sample from the
subject is greater than the control level of miR-24-1 gene product.
30. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. reverse transcribing at least miR-24-1 gene product from a test sample from
a
subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least miR-24-1 target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miR-24-1 RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, colon
cancer
if the level of at least one miR gene product in the hybridization profile
from
the test sample is greater than the control level of miR-24-1 gene product.
31. A method of diagnosing whether a subject has, or is at risk for
developing, lung
cancer, comprising:
a. comparing the level of at least one miR gene product in a test sample
from a
subject to a control level of at least one corresponding miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, lung
cancer,
if the level of the at least one miR gene product in the test sample from the
subject is greater than a control level of the corresponding miR gene product,
wherein the miR gene product is selected from the group consisting of: miR-
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21; miR-205; miR-200b; miR-9-1; miR-210; miR-148; miR-141; miR-132;
miR-215; miR-128b; let-7g; miR-16-2; miR-129-1/2 prec; miR-126; miR-
142-as; miR-30d; miR-30a-5p; miR-7-2; miR-199a-1; miR-127; miR-34a
prec; miR-34a; miR-136; miR-202; miR-196-2; miR-199a-2; let-7a-2; miR-
124a-1; miR-149; miR-17-5p; miR-196-1 prec; miR-10a; miR-99b prec;
miR-196-1; miR-199b; miR-191; miR-195; and miR-155.
32. A method of diagnosing whether a subject has, or is at risk for
developing, lung
cancer, comprising:
a. reverse transcribing at least one miR gene product from a test sample from
the subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least one miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, lung
cancer if
the level of at least one miR gene product in the test sample is greater than
the control level of one miR gene product, wherein the miR gene product is
selected from the group consisting of: miR-21; miR-205; miR-200b; miR-9-
1; miR-210; miR-148; miR-141; miR-132; miR-215; miR-128b; let-7g; miR-
16-2; miR-129-1/2 prec; miR-126; miR-142-as; miR-30d; miR-30a-5p; miR-
7-2; miR-199a-1; miR-127; miR-34a prec; miR-34a; miR-136; miR-202;
miR-196-2; miR-199a-2; let-7a-2; miR-124a-1; miR-149; miR-17-5p; miR-
196-1 prec; miR-10a; miR-99b prec; miR-196-1; miR-199b; miR-191; miR-
195; and miR-155.
33. A method of diagnosing whether a subject has, or is at risk for
developing, pancreatic
cancer, comprising:
a. comparing the level of at least one miR gene product in a test
sample from a
subject to a control level of at least one corresponding miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer, if the level of the at least one miR gene product in the test sample
from the subject is greater than a control level of the corresponding miR gene
product, wherein the miR gene product is selected from the group consisting
of: miR-103-2; miR-103-1; miR-24-2; miR-107; miR-100; miR-125b-2; miR-
125b-1; miR-24-1; miR-191; miR-23a; miR-26a-1; miR-125a; miR-130a;
118

miR-26b; miR-145; miR-221; miR-126; miR-16-2; miR-146; miR-214; miR-
99b; miR-128b; miR-155; miR-29b-2; miR-29a; miR-25; miR-16-1; miR-
99a; miR-224; miR-30d; miR-92-2; miR-199a-1; miR-223; miR-29c; miR-
30b; miR-129-1/2; miR-197; miR-17-5p; miR-30c; miR-7-1; miR-93-1; miR-
140; miR-30a-5p; miR-132; miR-181b-1; miR-152 prec; miR-23b; miR-20a;
miR-222; miR-27a; miR-92-1; miR-21; miR-129-1/2 prec; miR-150; miR-32;
miR-106a; and miR-29b-1.
34. A method of diagnosing whether a subject has, or is at risk for
developing, pancreatic
cancer, comprising:
a. reverse transcribing at least one miR gene product from a test sample from
the subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least one miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least one miR gene product in the test sample is
greater than the control level of one miR gene product, wherein the miR gene
product is selected from the group consisting of: miR-103-2; miR-103-1;
miR-24-2; miR-107; miR-100; miR-125b-2; miR-125b-1; miR-24-1; miR-
191; miR-23a; miR-26a-1; miR-125a; miR-130a; miR-26b; miR-145; miR-
221; miR-126; miR-16-2; miR-146; miR-214; miR-99b; miR-128b; miR-155;
miR-29b-2; miR-29a; miR-25; miR-16-1; miR-99a; miR-224; miR-30d; miR-
92-2; miR-199a-1; miR-223; miR-29c; miR-30b; miR-129-1/2; miR-197;
miR-17-5p; miR-30c; miR-7-1; miR-93-1; miR-140; miR-30a-5p; miR-132;
miR-181b-1; miR-152 prec; miR-23b; miR-20a; miR-222; miR-27a; miR-92-
1; miR-21; miR-129-1/2 prec; miR-150; miR-32; miR-106a; and miR-29b-1.
35. A method of diagnosing whether a subject has, or is at risk for
developing, pancreatic
cancer, comprising:
a. comparing the level of at least miR-100 gene product in a test sample from
a
subject to a control level of at least one miR-100 gene product; and
b. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer, if the level of at least miR-100 gene product in the test sample from
the subject is greater than the control level of miR-100 gene product.
119

36. A method of diagnosing whether a subject has, or is at risk for
developing, pancreatic
cancer, comprising:
a. reverse transcribing at least miR-100 gene product from a test sample from
a
subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least miR-100 target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miR-100 RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer if the level of at least one miR gene product in the hybridization
profile from the test sample is greater than the control level of miR-100 gene
product.
37. A method of diagnosing whether a subject has, or is at risk for
developing, pancreatic
cancer, comprising:
a. comparing the level of at least miR-155 gene product in a test sample from
a
subject to a control level of at least one miR-155 gene product; and
b. diagnosing whether the subject has, or is at risk for developing,
pancreatic
cancer, if the level of at least miR-155 gene product in the test sample from
the subject is less than the control level of miR-155 gene product.
38. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. comparing the level of at least one miR gene product in a test sample from
a
subject to a control level of at least one corresponding miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, prostate
cancer, if the level of the at least one miR gene product in the test sample
from the subject is greater than a control level of the corresponding miR gene
product, wherein the miR gene product is selected from the group consisting
of: let-7d; miR-128a prec; miR-195; miR-203; let-7a-2 prec; miR-34a; miR-
20a; miR-218-2; miR-29a; miR-25; miR-95; miR-197; miR-135-2; miR-187;
miR-196-1; miR-148; miR-191; miR-21; let-7i; miR-198; miR-199a-2; miR-
30c; miR-17-5p; miR-92-2; miR-146; miR-181b-1 prec; miR-32; miR-206;
miR-184 prec; miR-29a prec; miR-29b-2; miR-149; miR-181b-1; miR-196-1;
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miR-93-1; miR-223; miR-16-1; miR-101-1 prec; miR-124a-1; miR-26a-1;
miR-214; miR-27a; miR-24-1; miR-106a; and miR-199a-1.
39. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. reverse transcribing at least one miR gene product from a test sample from
the subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least one miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, prostate
cancer if the level of at least one miR gene product in the test sample is
greater than the control level of one miR gene product, wherein the miR gene
product is selected from the group consisting of: let-7d; miR-128a prec; miR-
195; miR-203; let-7a-2 prec; miR-34a; miR-20a; miR-218-2; miR-29a; miR-
25; miR-95; miR-197; miR-135-2; miR-187; miR-196-1; miR-148; miR-191;
miR-21; let-7i; miR-198; miR-199a-2; miR-30c; miR-17-5p; miR-92-2; miR-
146; miR-181b-1 prec; miR-32; miR-206; miR-184 prec; miR-29a prec; miR-
29b-2; miR-149; miR-181b-1; miR-196-1; miR-93-1; miR-223; miR-16-1;
miR-101-1 prec; miR-124a-1; miR-26a-1; miR-214; miR-27a; miR-24-1;
miR-106a; and miR-199a-1.
40. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. comparing the level of at least let-7d gene product in a test sample from a
subject to a control level of at least one let-7d gene product; and
b. diagnosing whether the subject has, or is at risk for developing, prostate
cancer, if the level of at least let-7d gene product in the test sample from
the
subject is greater than the control level of let-7d gene product.
41. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. reverse transcribing at least let-7d gene product from a test sample from a
subject to provide at least one corresponding miR gene product target
oligonucleotides;
121

b. hybridizing at least let-7d target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding let-7d RNA specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, prostate
cancer if the level of at least one miR gene product in the hybridization
profile from the test sample is greater than the control level of let-7d gene
product.
42. A method of diagnosing whether a subject has, or is at risk for
developing, stomach
cancer, comprising:
a. comparing the level of at least one miR gene product in a test
sample from a
subject to a control level of at least one corresponding miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, stomach
cancer, if the level of the at least one miR gene product in the test sample
from the subject is greater than a control level of the corresponding miR gene
product, wherein the miR gene product is selected from the group consisting
of: miR-223; miR-21; miR-218-2; miR-103-2; miR-92-2; miR-25; miR-136;
miR-191; miR-221; miR-125b-2; miR-103-1; miR-214; miR-222; miR-212
prec; miR-125b-1; miR-100; miR-107; miR-92-1; miR-96; miR-192; miR-
23a; miR-215; miR-7-2; miR-13 8-2; miR-24-1; miR-99b; miR-33b; and miR-
24-2.
43. A method of diagnosing whether a subject has, or is at risk for
developing, stomach
cancer, comprising:
a. reverse transcribing at least one miR gene product from a test sample from
the subject to provide at least one corresponding miR gene product target
oligonucleotides;
b. hybridizing at least one miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least one
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to at least one control
hybridization profile; and
d. diagnosing whether the subject has, or is at risk for developing, stomach
cancer if the level of at least one miR gene product in the test sample is
greater than the control level of one miR gene product, wherein the miR gene
122

product is selected from the group consisting of: miR-223; miR-21; miR-218-
2; miR-103-2; miR-92-2; miR-25; miR-136; miR-191;. miR-221; miR-125b-
2; miR-103-1; miR-214; miR-222; miR-212 prec; miR-125b-1; miR-100;
miR-107; miR-92-1; miR-96; miR-192; miR-23a; miR-215; miR-7-2; miR-
138-2; miR-24-1; miR-99b; miR-33b; and miR-24-2.
44. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. comparing the level of at least miR-146 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-146 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer,
if the level of the at least one miR-146 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the corresponding miR-146 gene product and at least
one additional miR gene product.
45. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. comparing the level of at least miR-29b-2 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-29b-2 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer,
if the level of the at least one miR-29b-2 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the corresponding miR-29b-2 gene product and at least
one additional miR gene product.
46. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. comparing the level of at least miR-21 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-21 gene product and at least one additional
miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer,
if the level of the at least one miR-21 gene product and at least one
additional
miR gene product in the test sample from the subject is greater than a control
123

level of miR-21 gene product and less than a control level of at least one
corresponding additional miR gene product.
47. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. comparing the level of at least miR-29b-2 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-29b-2 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer,
if the level of the at least one miR-29b-2 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of miR-29b-2 gene product and less than a control level
of at least one corresponding additional miR gene product.
48. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. comparing the level of at least miR-146 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-146 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, breast
cancer,
if the level of the at least one miR-146 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of miR-146 gene product and less than a control level of
at least one corresponding additional miR gene product.
49. A method of diagnosing whether a subject has, or is at risk for
developing, breast
cancer, comprising:
a. reverse transcribing at least two miR gene products from a test sample from
the subject to provide at least two corresponding miR gene product target
oligonucleotides;
b. hybridizing at least two miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least two
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to a control hybridization
profile for at least the two miR gene products; and
124

d. diagnosing whether the subject has, or is at risk for developing, breast
cancer
if the level of at least two gene products in the test sample is greater than
the
control level of the two gene products, wherein the two miR gene products
are gene products of the miRs selected from the group consisting of: miR-21;
miR-29b-2; miR-146; miR-125b-2; miR-125b-1; miR-10b; miR-145; miR-
181a; miR-140; miR-213; miR-29a prec; miR-181b-1; miR-199b; miR-29b-
1; miR-130a; miR-155; let-7a-2; miR-205; miR-29c; miR-224; miR-100;
miR-31; miR-30c; miR-17-5p; miR-210; miR-122a; and miR-16-2.
50. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. comparing the level of at least miR-24-1 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-24-1 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, colon
cancer,
if the level of the at least one miR-24-1 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-24-1 gene product and at least one
corresponding additional miR gene product.
51. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. comparing the level of at least miR-29b-2 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-29b-2 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, colon
cancer,
if the level of the at least one miR-29b-2 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-29b-2 gene product and at least one
corresponding additional miR gene product.
52. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. comparing the level of at least miR-20a gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-20a gene product and at least one
additional miR gene product; and
125

b. diagnosing whether the subject has, or is at risk for developing, colon
cancer,
if the level of the at least one miR-20a gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-20a gene product and at least one
corresponding additional miR gene product.
53. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. comparing the level of at least miR-10a gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-10a gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, colon
cancer,
if the level of the at least one miR-10a gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-10a gene product and at least one
corresponding additional miR gene product.
54. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. comparing the level of at least miR-32 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-32 gene product and at least one additional
miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, colon
cancer,
if the level of the at least one miR-32 gene product and at least one
additional
miR gene product in the test sample from the subject is greater than a control
level of the miR-32 gene product and at least one corresponding additional
miR gene product.
55. A method of diagnosing whether a subject has, or is at risk for
developing, colon
cancer, comprising:
a. reverse transcribing at least two miR gene products from a test sample from
the subject to provide at least two corresponding miR gene product target
oligonucleotides;
b. hybridizing at least two miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least two
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
126

c. comparing the test sample hybridization profile to a control hybridization
profile for at least the two miR gene products; and
d. diagnosing whether the subject has, or is at risk for developing, colon
cancer
if the level of at least two gene products in the test sample is greater than
the
control level of the two gene products, wherein the two miR gene products
are gene products of the miRs selected from the group consisting of: miR-24-
1; miR-29b-2; miR-20a; miR-10a; miR-32; miR-203; miR-106a; miR-17-5p;
miR-30c; miR-223; miR-126; miR-128b; miR-21; miR-24-2; miR-99b prec;
miR-155; miR-213; miR-150; miR-107; miR-191; miR-221; and miR-9-3.
56. A method of diagnosing whether a subject has, or is at risk for
developing, lung
cancer, comprising:
a. comparing the level of at least miR-205 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-205 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, lung
cancer,
if the level of the at least one miR-205 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-205 gene product and at least one
corresponding additional miR gene product.
57. A method of diagnosing whether a subject has, or is at risk for
developing, lung
cancer, comprising:
a. comparing the level of at least miR-200b gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-200b gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, lung
cancer,
if the level of the at least one miR-200b gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-200b gene product and at least one
corresponding additional miR gene product.
58. A method of diagnosing whether a subject has, or is at risk for
developing, lung
cancer, comprising:
a. comparing the level of at least miR-9-1 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
127

of at least one corresponding miR-9-1 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, lung
cancer,
if the level of the at least one miR-9-1 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-9-1 gene product and at least one
corresponding additional miR gene product.
59. A method of diagnosing whether a subject has, or is at risk for
developing, lung
cancer, comprising:
a. comparing the level of at least miR-210 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-210 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, lung
cancer,
if the level of the at least one miR-210 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-210 gene product and at least one
corresponding additional miR gene product.
60. A method of diagnosing whether a subject has, or is at risk for
developing, lung
cancer, comprising:
a. comparing the level of at least miR-148 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one miR-148 gene product and at least one corresponding
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, lung
cancer,
if the level of the at least one miR-148 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-148 gene product and at least one
corresponding additional miR gene product.
61. A method of diagnosing whether a subject has, or is at risk for
developing, lung
cancer, comprising:
a. reverse transcribing at least two miR gene products from a test sample from
the subject to provide at least two corresponding miR gene product target
oligonucleotides;
b. hybridizing at least two miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least two
128

corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to a control hybridization
profile for at least the two miR gene products; and
d. diagnosing whether the subject has, or is at risk for developing, lung
cancer if
the level of at least two gene products in the test sample is greater than the
control level of the two gene products, wherein the two miR gene products
are gene products of the miRs selected from the group consisting of: miR-21;
miR-205; miR-200b; miR-9-1; miR-210; miR-148; miR-141; miR-132; miR-
215; miR-128b; let-7g; miR-16-2; miR-129-1/2 prec; miR-126; miR-142-as;
miR-30d; miR-30a-5p; miR-7-2; miR-199a-1; miR-127; miR-34a prec; miR-
34a; miR-136; miR-202; miR-196-2; miR-199a-2; let-7a-2; miR-124a-1;
miR-149; miR-17-5p; miR-196-1 prec; miR-10a; miR-99b prec; miR-196-1;
miR-199b; miR-191; miR-195; and miR-155.
62. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. comparing the level of at least let-7d gene product and at least one
additional
miR gene product in a test sample from a subject to a control level of at
least
one corresponding let-7d gene product and at least one additional miR gene
product; and
b. diagnosing whether the subject has, or is at risk for developing, prostate
cancer, if the level of the at least one let-7d gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the let-7d gene product and at least one corresponding
additional miR gene product.
63. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. comparing the level of at least miR-128a prec gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-128a prec gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, prostate
cancer, if the level of the at least one miR-128a prec gene product and at
least
one additional miR gene product in the test sample from the subject is greater
than a control level of the miR-128a prec gene product and at least one
corresponding additional miR gene product.
129

64. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. comparing the level of at least miR-195 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-195 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, prostate
cancer, if the level of the at least one miR-195 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-195 gene product and at least one
corresponding additional miR gene product.
65. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. comparing the level of at least miR-203 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-203 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, prostate
cancer, if the level of the at least one miR-203 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-203 gene product and at least one
corresponding additional miR gene product.
66. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
a. comparing the level of at least let-7a-prec gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding let-7a-prec gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, prostate
cancer, if the level of the at least one let-7a-prec gene product and at least
one
additional miR gene product in the test sample from the subject is greater
than a control level of the let-7a-prec gene product and at least one
corresponding additional miR gene product.
67. A method of diagnosing whether a subject has, or is at risk for
developing, prostate
cancer, comprising:
130

a. reverse transcribing at least two miR gene products from a test sample from
the subject to provide at least two corresponding miR gene product target
oligonucleotides;
b. hybridizing at least two miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least two
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to a control hybridization
profile for at least the two miR gene products; and
d. diagnosing whether the subject has, or is at risk for developing, prostate
cancer if the level of at least two gene products in the test sample is
greater
than the control level of the two gene products, wherein the two miR gene
products are gene products of the miRs selected from the group consisting of:
let-7d; miR-128a prec; miR-195; miR-203; let-7a-2 prec; miR-34a; miR-20a;
miR-218-2; miR-29a; miR-25; miR-95; miR-197; miR-135-2; miR-187; miR-
196-1; miR-148; miR-191; miR-21; let-7i; miR-198; miR-199a-2; miR-30c;
miR-17-5p; miR-92-2; miR-146; miR-181b-1 prec; miR-32; miR-206; miR-
184 prec; miR-29a prec; miR-29b-2; miR-149; miR-181b-1; miR-196-1;
miR-93-1; miR-223; miR-16-1; miR-101-1 prec; miR-124a-1; miR-26a-1;
miR-214; miR-27a; miR-24-1; miR-106a; and miR-199a-1.
68. A method of diagnosing whether a subject has, or is at risk for
developing, stomach
cancer, comprising:
a. comparing the level of at least miR-223 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-223 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, stomach
cancer, if the level of the at least one miR-223 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-223 gene product and at least one
corresponding additional miR gene product.
69. A method of diagnosing whether a subject has, or is at risk for
developing, stomach
cancer, comprising:
a. comparing the level of at least miR-103-2 prec gene product and at least
one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-103-2 gene product and at least one
additional miR gene product; and
131

b. diagnosing whether the subject has, or is at risk for developing, stomach
cancer, if the level of the at least one miR-103-2 gene product and at least
one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-103-2 gene product and at least one
corresponding additional miR gene product.
70. A method of diagnosing whether a subject has, or is at risk for
developing, stomach
cancer, comprising:
a. comparing the level of at least miR-92-2 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-92-2 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, stomach
cancer, if the level of the at least one miR-92-2 gene product and at least
one
additional miR gene product in the test sample from the subject is greater
than a control level of the miR-92-2 gene product and at least one
corresponding additional miR gene product.
71. A method of diagnosing whether a subject has, or is at risk for
developing, stomach
cancer, comprising:
a. comparing the level of at least miR-25 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-25 gene product and at least one additional
miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, stomach
cancer, if the level of the at least one miR-25 gene product and at least one
additional miR gene product in the test sample from the subject is greater
than a control level of the corresponding miR-25 gene product and at least
one additional miR gene product.
72. A method of diagnosing whether a subject has, or is at risk for
developing, stomach
cancer, comprising:
a. comparing the level of at least miR-218-2 gene product and at least one
additional miR gene product in a test sample from a subject to a control level
of at least one corresponding miR-218-2 gene product and at least one
additional miR gene product; and
b. diagnosing whether the subject has, or is at risk for developing, stomach
cancer, if the level of the at least one miR-218-2 gene product is less than a
control level of miR-218-2 gene product and the least one additional miR
132

gene product in the test sample is greater than a control level of the
corresponding at least one additional miR gene product.
73. A method of diagnosing whether a subject has, or is at risk for
developing, stomach
cancer, comprising:
a. reverse transcribing at least two miR gene products from a test sample from
the subject to provide at least two corresponding miR gene product target
oligonucleotides;
b. hybridizing at least two miR target oligodeoxynucleotides to a microarray
comprising miRNA-specific probe oligonucleotides that include at least two
corresponding miRNA-specific probe oligonucleotides to provide a
hybridization profile for the test sample;
c. comparing the test sample hybridization profile to a control hybridization
profile for at least the two miR gene products; and
d. diagnosing whether the subject has, or is at risk for developing, stomach
cancer if the level of at least two gene products in the test sample is
greater
than the control level of the two gene products, wherein the two miR gene
products are gene products of the miRs selected from the group consisting of:
miR-223; miR-21; miR-218-2; miR-103-2; miR-92-2; miR-25; miR-136;
miR-191; miR-221; miR-125b-2; miR-103-1; miR-214; miR-222; miR-212
prec; miR-125b-1; miR-100; miR-107; miR-92-1; miR-96; miR-192; miR-
23a; miR-215; miR-7-2; miR-138-2; miR-24-1; miR-99b; miR-33b; and miR-
24-2.
74. A method of diagnosing whether a subject has a solid cancer comprising:
a. measuring the level of at least a first miR-17-5p gene product in a test
sample
from the subject,
b. wherein an alteration in the level of the first miR-17-5p gene product in
the
test sample, relative to the level of the first miR-17-5p gene product in a
control sample,
is indicative of the subject having a solid cancer selected from the group
consisting of breast, colon, lung, pancreas and prostate cancer.
75. A method of diagnosing whether a subject has breast cancer, comprising:
measuring the level of at least one miR gene product selected from the group
consisting of miR-125b-2, miR-125b-1, miR-10b, miR-181a, miR-140, miR-213,
miR-29a prec, miR-199b, miR-29b-1, miR-130a, miR-155, let-7a-2, miR-29c, miR-
133

224, miR-31, miR-122a, miR-16-2, miR-30c, miR-17-5p, miR-210, miR-29b-2,
miR-146, miR-181b-1, and combinations thereof in a test sample from the
subject,
wherein an alteration in the level of the miR gene product in the test sample,
relative to the level of the corresponding miR gene product in a control
sample, is
indicative of the subject having breast cancer.
76. A method of diagnosing whether a subject has colon cancer, comprising:
measuring the level of at least one miR gene product selected from the group
consisting of miR-29b-2, miR-126*, miR-99b prec, miR-213, miR-150, miR-191,
miR-221, miR-9-3, miR-24-1, miR-20a, miR-32, miR-17-5p, miR-30c, miR-128b,
miR-24-2, miR-155, miR-107 and combinations thereof in a test sample from the
subject,
wherein an alteration in the level of the miR gene productin the test sample,
relative to the level of the corresponding miR gene product in a control
sample, is
indicative of the subject having colon cancer.
77. A method of diagnosing whether a subject has lung cancer, comprising:
measuring the level of at least one miR gene product selected from the group
consisting of miR-9-1, miR-148, miR-141, miR-132, miR-215, let-7g, miR-16-2,
miR-129-1/2 prec, miR-126*, miR-142-as, miR-30d, miR-30a-5p, miR-7-2, miR-
127, miR-34a prec, miR-34a, miR-136, miR-202, miR-196-2, miR-199a-2, let-7a-2,
miR-124a-1, miR-149, miR-196-1 prec, miR-10a, miR-99b prec, miR-196-1, miR-
199b, miR-191, miR-155, miR-210, miR-128b, miR-199a-1, miR-17-5p and
combinations thereof in a test sample from the subject,
wherein an alteration in the level of the miR gene products in the test
sample,
relative to the level of the corresponding miR gene product in a control
sample, is
indicative of the subject having lung cancer.
78. A method of diagnosing whether a subject has pancreatic cancer,
comprising:
measuring the level of at least one miR gene product selected from the group
consisting of miR-103-2, miR-103-1, miR-107, miR-100, miR-125b-2, miR-125b-1,
miR-191, miR-23a, miR-26a-1, miR-125a, miR-130a, miR-26b, miR-145, miR-221,
miR-126*, miR-16-2, miR-99b, miR-29a, miR-16-1, miR-99a, miR-224, miR-30d,
134

miR-29c, miR-30b, miR-129-1/2, miR-197, miR-30c, miR-7-1, miR-93-1, miR-140,
miR-30a-5p, miR-132, miR-152 prec, miR-23b, miR-222, miR-27a, miR-92-1, miR-
21, miR-129-1/2 prec, miR-150, miR-29b-1, miR-128b, miR-155, miR-29b-2, miR-
25, miR-92-2, miR-199a-1, miR-223, miR-17-5p, miR-181b-1, miR-20a, miR-32,
miR-106a, miR-146, miR-214, miR-24-1, miR-24-2 and combinations thereof in a
test sample from the subject,
wherein an alteration in the level of the miR gene product in the test sample,
relative to the level of the corresponding miR gene product in a control
sample, is
indicative of the subject having pancreatic cancer.
79. A method of diagnosing whether a subject has prostate cancer, comprising:
measuring the level of at least one miR gene product selected from the group
consisting of let-7d, miR-128a prec, miR-195, miR-203, let-7a-2 prec, miR-34a,
miR-29a, miR-95, miR-197, miR-135-2, miR-187, miR-196-1, miR-148, let-7i, miR-
198, miR-199a-2, miR-181b-1 prec, miR-206, miR-184 prec, miR-29a prec, miR-
149, miR-196-1 prec, miR-93-1, miR-223, miR-16-1, miR-101-1, miR-124a-1, miR-
26a-1, miR-214, miR-27a, miR-24-1, miR-106a, miR-199a-1, miR-20a, miR-218-2,
miR-25, miR-191, miR-30c, miR-17-5p, miR-92-2, miR-146, miR-32, miR-29b-2,
miR-181b-1 and combinations thereof in a test sample from the subject,
wherein an alteration in the level of the miR gene product in the test sample,
relative to the level of the corresponding miR gene product in a control
sample is
indicative of the subject having prostate cancer.
80. A method of diagnosing whether a subject has stomach cancer, comprising:
measuring the level of at least one miR gene product selected from the group
consisting of miR-223, miR-21, miR-103-2, miR-136, miR-125b-2, miR-103-1,
miR-222, miR-212 prec, miR-125b-1, miR-100, miR-92-1, miR-96, miR-192, miR-
23a, miR-215, miR-7-2, miR-138-2, miR-99b, miR-33b, miR-218-2, miR-92-2, miR-
25, miR-191, miR-221, miR-214, miR-107, miR-24-1, miR-24-2 and combinations
thereof in a test sample from the subject,
wherein an alteration in the level of the miR gene product in the test sample,
relative to the level of the corresponding miR gene product in a control
sample, is
indicative of the subject having stomach cancer.
135

81. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-199a-1 gene product in a test
sample from the subject,
wherein an alteration in the level of the first miR-199a-1 gene product in the
test sample, relative to the level of the first miR-199a-1 gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of lung, pancreas and prostate cancer.
82. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-128b gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-128b gene product in the
test sample, relative to the level of the first miR-128b gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of colon, lung and pancreas cancer.
83. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-223 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-223 gene product in the
test
sample, relative to the level of the first miR-223 gene product in a control
sample, is
indicative of the subject having a solid cancer selected from the group
consisting of
colon, pancreas, prostate and stomach cancer; and
wherein, when the cancer is colon cancer, at least one additional miR gene
product is used to diagnose colon cancer, the at least one additional miR gene
product being selected from the group consisting of: miR-21, miR-17-5p, miR-
191,
miR-29b-2, miR-128b, miR-24-1, miR-24-2, miR-155, miR-20a, miR-107, miR-32,
miR-30c, miR-221, miR-106a, miR-10a, miR-203, miR-126*, miR-24-2, miR-
99b prec, miR-213, miR-150, miR-9-3 and combinations thereof.
84. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-29b-2 gene product in a test
sample
136

from the subject,
wherein an alteration in the level of the first miR-29b-2 gene product in the
test sample, relative to the level of the first miR-29b-2 gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of breast, colon, pancreas and prostate cancer.
85. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-24-1 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-24-1 gene product in the
test sample, relative to the level of the first miR-24-1 gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of colon, pancreas and stomach cancer.
86. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-24-2 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-24-2 gene product in the
test sample, relative to the level of the first miR-24-2 gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of colon, pancreas and stomach cancer.
87. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-146 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-146 gene product in the
test
sample, relative to the level of the first miR-146 gene product in a control
sample, is
indicative of the subject having a solid cancer selected from the group
consisting of
breast, pancreas and prostate cancer.
88. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-155 gene product in a test sample
from the subject,
137

wherein an alteration in the level of the first miR-155 gene product in the
test
sample, relative to the level of the first miR-155 gene product in a control
sample, is
indicative of the subject having a solid cancer selected from the group
consisting of
breast, colon, lung and pancreas cancer.
89. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-181b-1 gene product in a test
sample from the subject,
wherein an alteration in the level of the first miR-181b-1 gene product in the
test sample, relative to the level of the first miR-181b-1 gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of breast, pancreas and prostate cancer.
90. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-20a gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-20a gene product in the
test
sample, relative to the level of the first miR-20a gene product in a control
sample, is
indicative of the subject having a solid cancer selected from the group
consisting of
colon, pancreas and prostate cancer.
91. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-107 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-107 gene product in the
test
sample, relative to the level of the first miR-107 gene product in a control
sample, is
indicative of the subject having a solid cancer selected from the group
consisting of
colon, pancreas and stomach cancer.
92. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-32 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-32 gene product in the
test
sample, relative to the level of the first miR-32 gene product in a control
sample, is
138

indicative of the subject having a solid cancer selected from the group
consisting of
colon, pancreas and prostate cancer.
93. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-92-2 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-92-2 gene product in the
test sample, relative to the level of the first miR-92-2 gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of pancreas, prostate and stomach cancer.
94. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-214 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-214 gene product in the
test
sample, relative to the level of the first miR-214 gene product in a control
sample,
is indicative of the subject having a solid cancer selected from the group
consisting
of pancreas, prostate and stomach cancer.
95. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-30c gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-30c gene product in the
test
sample, relative to the level of the first miR-30c gene product in a control
sample, is
indicative of the subject having a solid cancer selected from the group
consisting of
colon, pancreas and prostate cancer.
96. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-25 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-25 gene product in the
test
sample, relative to the level of the first miR-25 gene product in a control
sample, is
indicative of the subject having a solid cancer selected from the group
consisting of
139

pancreas, prostate and stomach cancer.
97. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-221 gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-221 gene product in the
test
sample, relative to the level of the first miR-221 gene product in a control
sample,
is indicative of the subject having a solid cancer selected from the group
consisting
of colon, pancreas and stomach cancer.
98. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-106a gene product in a test sample
from the subject,
wherein an alteration in the level of the first miR-106a gene product in the
test sample, relative to the level of the first miR-106a gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of colon, pancreas and prostate cancer; and
wherein when the cancer is colon cancer, at least one additional miR gene
product used to diagnose colon cancer, the at least a second miR gene product
being
selected from the group consisting of: miR-21, miR-17-5p, miR-191, miR-29b-2,
miR-223, miR-128b, miR-24-1, miR-24-2, miR-155, miR-20a, miR-107, miR-32,
miR-30c, miR-221, miR-10a, miR-203, miR-126*, miR-24-2, miR-99b prec, miR-
213, miR-150, miR-9-3 and combinations thereof,
and if miR-21 is selected, further selecting at least a third miR gene product
selected from: miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-24-1,
miR-24-2, miR-155, miR-20a, miR-107, miR-32, miR-30c, miR-221, miR-10a,
miR-203, miR-126*, miR-24-2, miR-99b prec, miR-213, miR-150, miR-9-3 and
combinations thereof.
99. A method of diagnosing whether a subject has a solid cancer,
comprising:
measuring the level of at least a first miR-210 gene product in a test sample
from the subject,
140

wherein an alteration in the level of the first miR-210 gene product in the
test sample, relative to the level of the first miR-210 gene product in a
control
sample, is indicative of the subject having a solid cancer selected from the
group
consisting of breast or lung cancer.
100. A method of diagnosing whether a subject has a solid cancer comprising:
measuring the level of at least a first miR-218-2 gene product in a test
sample
from the subject,
wherein an alteration in the level of the first miR-218 gene product in the
test
sample, relative to the level of the first miR-218 gene product in a control
sample,
is indicative of the subject having a solid cancer selected from the group
consisting
of colon, pancreas, prostate and stomach cancer.
101. A method of diagnosing whether a subject has breast cancer, comprising:
measuring in a test sample from the subject the levels of a group of miR gene
products consisting of miR125-b-1, miR-125b-2, miR-145 and miR-21,
wherein an alteration in the levels of the group of miR gene products in the
test sample, relative to the levels of the corresponding miR gene products in
a
control sample, is indicative of the subject having breast cancer; and
wherein the alteration in the levels of the group of miR gene products
comprises:
i) an increase in the level of miR-21 gene product in the test
sample, relative to the control level of the corresponding gene product, and
ii) a decrease in the level of miR-125b-1, miR-125b-2 and miR-
145 gene product in the test sample, relative to the control level of the
corresponding gene products.
102. A method of diagnosing whether a subject has pancreatic cancer,
comprising:
measuring in a test sample from the subject the levels of a group of miR gene
products consisting of miR-103-1, miR-103-2, miR-155 and miR-204,
wherein an alteration in the levels of the group of miR gene products in the
test sample, relative to the levels of the corresponding miR gene products in
a
control sample, is indicative of the subject having pancreatic cancer; and
141

wherein the alteration in the levels of the group of miR gene products
comprises:
iii) an increase in the level of the miR-103-1, miR-103-2 and miR-
214 gene products in the test sample, relative to the control level of the
corresponding gene products, and
iv) a decrease in the level of miR-155 gene product in the test
sample, relative to the control level of the corresponding gene product.
142

Description

CA 02811486 2013-03-26
MicroRNA-BASED METHODS AND COMPOSITIONS
FOR THE DIAGNOSIS AND TREATMENT OF SOLID CANCERS
This application is a divisional of Canadian Patent Application No. 2,633,754,
filed
on January 3, 2007. The claims of this application are directed generally to
microRNA-
based methods to diagnose if a patient has, or is risk for developing, various
solid cancers.
The retention of any clauses or features which may be more particularly
related to
the parent application or a separate divisional thereof should not be regarded
as rendering
the teachings and claiming ambiguous or inconsistent with the subject matter
defined in the
claims of the divisional application presented herein when seeking to
interpret the scope
thereof and the basis in this disclosure for the claims recited herein.
BACKGROUND OF THE INVENTION
Cancer, the uncontrolled growth of malignant cells, is a major health problem
of
the modern medical era and is one of the leading causes of death in developed
countries. In the United States, one in four deaths is caused by cancer
(Jemal, A. et al.,
CA Cancer I Clin. 52:23-47 (2002)). Among cancers, those that arise from
organs and
solid tissues, known as solid cancers (e.g., colon cancer, lung cancer, breast
cancer,
stomach cancer, prostate cancer, pancreatic cancer) are among the most-
commonly
identified human cancers.
For example, prostate cancer is the most frequently diagnosed noncutaneous
malignancy among men in industrialized countries, and, in the United States, 1
in 8
men will develop prostate cancer during his life (Simard, J. et al.,
Endocrinology
143(6):2029-40(2002)). The incidence of prostate cancer has dramatically
increased
over the last decades and prostate cancer is now a leading cause of death in
the United
States and Western Europe (Preschel, R.E. and J.W. Colberg, Lancet 4:233-41
(2003);
Nelson, W.G. etal., N. Engl. I Med. 349(4):366-81 (2003)). An average 40%
reduction in life expectancy affects males with prostate cancer. If detected
early, prior
to metastasis and local spread beyond the capsule, prostate cancer can often
time be
cured (e.g., using surgery). However, if diagnosed after spread and metastasis
from the
prostate, prostate cancer is typically a fatal disease with low cure rates.
While prostate-
specific antigen (PSA)-based screening has aided early diagnosis of prostate
cancer, it
is neither highly sensitive nor specific (Punglia etal., N. Engl. I Med.
349(4):335-42
1

- CA 02811486 2013-03-26
=
(2003)). This means that a high percentage of false negative and false
positive
diagnoses are associated with the test. The consequencesare both many
instances of.
missed cancers and unnecessai follow-up biopsies for those without cancer.=
Breast cancer remains the second leading cause of cancer-related deaths in
= 5 women, affecting more than 180,000 women in the United States
each year. For"
women in North America, the life-time odds of getting breast cancer are now
one in
eight. Although the discovery of BRCA1 and BRCA2 were important steps in
identifying key genetic factors involved in breast cancer, it has become clear
that
mutations in BRCA1 and BRCA2 account for only a fraction of inherited
susceptibility
to breast cancer (Nathanson, KL., et al., Human Mol. Gen. 10(7):715-720
(2001);
Anglican Breast Cancer Study Group. Br. J Cancer 83(70):1301-08 (2000); and
Syrjakosld, K., et al., J Natl. Cancer Inst. .92:1529-31 (2000)). Despite
considerable
= research into therapies for breast cancer, breast cancer remains
difficult to diagnose and
treat effectively; and the high mortality observed in breast cancer patients
indicates that
improvements are needed in the diagnosis, treatment and prevention of the
disease.
Excluding skin cancer, colorectal cancer is thelhird most frequently diagnosed
cancer in the United States and Canada (after huag. and breast in women, and
lung and
prostate in men). The American Cancer Society estimates that there will be
approximately 145,000 new cases of colorectal cancer diagnosed in the 'U.S. in
2005
(Cancer Facts and Figures 2005. Atlanta, GA: American Cancer Society, 2005,7Y.
_
Colorectal cancer is the second leading cause of cancer death among men and
women =
in the United States and Canada (after lung cancer).
.The annual incidence of pancreatic cancer is nearly equivalent to the atunial
Mortality, estimated to be 31,860 and 31,270, respectively, in the U.S." in
2004 "(Cancer
F4cts and Figures 2004. Atlanta, GA: American Cancer Society, 2004a ). Padeats
with
_ = _
=
locally advanced and metastatic pancreatic cancer have poor prognoses, and
diagnosis
generally occurs too late for surgery or radiotherapy to be curative (Burr,
HA., et a?.,
The Oncologist ./0(3): 183-190, (2005)). ChemotheraPy can provide relief of
synaptams for some patients with advanced pancreatic cancer, but its impact on
survival
ha..4. been modest to date.
=
=
=
=

CA 02811486 2013-03-26
=
=
In the United States, more than 20,000 individuals are diagnosed with stomach
(gastric) cancer each year. The American Cancer Society estimates that there
will be
22,710 new cases of colorectal cancer diagnosed in the U.S. in 2004 (Cancer
Facts and
Figures 2004. Atlanta, GA: American Cancer Society, 2004.). Because
stomach cancer may occur without symptoms, it may be in advanced stages by the
time
the diagnosis is made. Treatment is then directed at making the patient more
comfortable and improving quality of life.
Lung cancer causes more deaths worldwide than any other form of cancer
(Goodman, G.E., Thorax 57:994-999 (2002)). In the United States, lung cancer
is the
primary cause of cancer death among both men and women. In 2002, the death
rate
from lung cancer was an estimated 134,900 deaths, exceeding the combined total
for
breast, prostate and colon cancer. Id. Lung cancer is also the leading cause
of cancer
death in all European countries, and numbers of lung cancer-related deaths are
rapidly
=
=
increasing in developing countries as well.
The five-year survival rate among all lung cancer patients, regardless of the
stage of disease at diagnosis, is only about 13%. This contrasts with a five-
year
survival rate of 46% among cases detected while the disease is still
localized.
However, only 16% of lung cancers are discovered before the disease has
spread. Early "
detection is difficult as clinical symptoms are often not observed until the
disease has
reached an advanced stage. Despite research into therapies for this and other
cancers,
lung cancer remains difficult to diagnose and treat effectively.
Clearly, the identification of markers and genes that are responsible for
susceptibility to particular forms of solid cancer (e.g., prostate cancer,
breast cancer,
lung cancer, stomach cancer, colon cancer, pancreatic cancer) is one of the
major
challenges facing oncology today. There is a need to identify means for the
early
detection of individuals that have a genetic susceptibility to cancer so that
more
=
aggressive screening and intervention regimens may be instituted for the early
detection
and treatment of cancer. Cancer genes may also reveal key molecular pathways
that
may be manipulated (e.g., using small or large molecule weight drugs) and may
lead to
more effective treatments regardless of the cancer stage when a particular
cancer is first
diagnosed.
3

CA 02811486 2013-03-26
MicroRNAs are a class of small, non-coding RNAs that control gene expression
by hybridizing to and triggering either translational repression or, less
frequently,
degradation of a messenger RNA (mRNA) target. The discovery and study of
miRNAs
has revealed miRNA-mediated gene regulatory mechanisms that play important
roles in
organismal development and various cellular processes, such as cell
differentiation, cell
growth and cell death (Cheng, A.M., et al., Nucleic Acids Res. 33:1290-1297
(2005)).
Recent studies suggest that aberrant expression of particular miRNAs may be
involved
in human diseases, such as neurological disorders (Ishizuka, A., et aL, Genes
Day.
/6:2497-2508 (2002)) and cancer. In particular, misexpression of miR-16-1
and/or
miR-15a has been found in human chronic lymphocytic leukemias (Calm, (l.A., et
al.,
Proc. Natl. Acad. Sci. U.S.A. 99:15524-15529 (2002)).
Clearly, there is a great need in the art for improved methods for detecting
and
treating solid cancers (e.g., prostate cancer, breast cancer, lung cancer,
stomach cancer,
colon cancer, pancreatic cancer). The present invention provides novel
niethods and
compositions for the diagnosis and treatment of solid cancers.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the identification of specific
miRNAs
that have altered expression levels in particular solid cancers.
Accordingly, the invention encompasses methods of diagnosing whether a
subject has, or is at risk for developing, a solid cancer. According to the
methods of the
invention, the level of at least one miR gene product in a test sample from
the subject is
compared to the level of a corresponding miR gene product in a control sample.
An
alteration (e.g., an increase, a decrease) in the leve.1 of the miR gene
product in the test
sample, relative to the level of a corresponding miR gene product in a control
sample,
is indicative of the subject either having, or being at risk for developing, a
solid cancer.
The solid cancer can be any cancer that arises from organs and solid tissues.
In certain
embodiments, the solid cancer is stomach cancer, breast cancer, pancreatic
cancer,
colon cancer, lung cancer or prostate cancer. In particular embodiments, the
solid
cancer is not breast cancer, lung cancer, prostate cancer, pancreatic cancer
or
gastrointestinal cancer.
4

CA 02811486 2013-03-26
In one embodiment, the at leaSt one miR gene product measured in the test
sample is selected from the group consisting of miR-21, miR-191, miR-17-5p and
combinations thereof. In another embodiment, the at least one miR gene product
measured in the test sample is selected from the group consisting of miR-21,
miR-17-
5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2,
miR-146, miR-155, miR-181b-1, miR-20a, miR.-107, miR-32, miR-92-2, miR-214,
miR-30c, miR-25, miR-221, miR-106a and combinations thereof.
In one embodiment, the solid cancer is breast cancer or lung cancer and the at
least one miR gene product measured in the test sample is selected from the
group
consisting of miR-210, miR-213 and a combination thereof.
In another embodiment, the solid cancer is colon cancer, stomach cancer,
prostate cancer or pancreas cancer and the at least one miR gene product
measured in
the test sample is miR-218-2.
In a certain embodiment, the solid cancer is breast cancer and the at least
one
miR gene product measured in the test sample is selected from the group
consisting of
trtiR-125b-1, miR-125b-2, miR-145, miR-21 and combinations thereof. In a
related
embodiment, the solid cancer is breast cancer and the at least one miR gene
product in
the test sample is selected from the group consisting of miR-21, miR-29b-2,
miR-146,
miR-125b-2, miR-125b-1, miR-10b, miR-145, miR-181a, miR-140, miR-213, miR-29a
prec, miR-181b-1, miR-199b, miR-29b-1, miR-130a, miR-155, let-7a-2, miR-205,
zniR-29c, miR-224, miR-100, miR-31, miR-30c, miR-17-5p, miR-210, miR-122a, miR-
16-2 and combinations thereof.
In another embodiment, the solid cancer is colon cancer and the at least one
miR gene product in the test sample is selected from the group consisting of
miR-24-1,
miR-29b-2, miR-20a, miR-10a, miR-32, miR-203, miR-106a, miR-17-5p, miR-30c,
'MR-223, miR-126*, miR-128b, milk-21, miR-24-2, miR-99b prec, miR-155, miR-
213,
miR-150, miR-107, miR-191, miR-221, miR-9-3 and combinations thereof.
In yet another embodiment, the solid cancer is lung cancer and the miR gene
product in the test sample is selected from the group consisting of miR-21,
miR-205,
miR-200b, miR-9-1, miR-210, miR-148, miR-141, miR-132, miR-215, miR-128b, let-
7g, miR-16-2, miR-129-1/2 prec, miR-126*, miR-142-as, miR-30d, miR-30a-5p, miR-
7-2, miR-199a-1, miR-127, miR-34a prec, miR-34a, miR-136, niiR-202, miR.-196-
2,
=
5
=

CA 02811486 2013-03-26
= miR-199a-2, let-7a-2, miR-124a-1, miR-149, miR-17-5p, miR-196-1 prec, miR-
10a,
miR-99b prec, miR-196-1, miR-199b, miR-191, miR-195, miR-155 and combinations
thereof.
In an additional embodiment, the solid cancer is pancreatic cancer and the at
least one miR gene product measured in the test sample is selected from the
group
consisting of miR-103-1, miR-103-2, miR-155, miR-2O4 and combinations thereof.
In
a related embodiment, the solid cancer is pancreatic cancer and the miR gene
product in
the test sample is selected from the group consisting of miR-103-2, miR-103-1,
miR-
24-2, miR-107, miR-100, miR-125b-2, miR-125b-1, miR-24-1, miR-191, miR-23a,
miR-26a-1, miR-125a, miR-130a, miR-26b, miR-145, miR-221, miR-126*,. rniR-16-
2,
miR-146, miR-214, miR-99b, tniR-128b, miR-155, miR-29b-2, miR-29a, miR-25,
miR-16-1, miR-99a, miR-224, miR-30d, miR-92-2, miR-199a-1, miR-223, rniR-29c,
miR-30b, miR-129-1/2, miR-197, miR-17-5p, miR-30c, miR-7-1, miR-93-1, miR-140,
miR-30a-5p, miR-132, miR-181b-1, miR-152 prec, miR-23b, miR-20a, miR-222, miR-
=
27a7 miR-92-1, miR-21, miR-129-1/2 prec, miR-150, miR-32, miR-106a, miR-29b-1
and combinations thereof.
In another embodiment, the solid cancer is prostate cancer and the miR gene
product in the test sample is selected from the group consisting of let-7d,
miR-128a
prec, miR-195, miR-203, let-7a-2 prec, miR-34a, miR-20a, miR-218-2, miR-29a,
rniR-
25, miR-95, miR-197, miR-135-2, miR-187, miR-196-1, miR-148, miR-191, miR-21,
let-7i, miR-198, miR-199a-2, miR-30c, miR-17-5p, miR-92-2, miR-146, miR-181b-1
prec, miR-32, miR-206, miR-184 prec, miR-29a prec, miR-29b-2, miR-149, miR-18
lb-
1, miR-196-1 prec, miR-93-1, miR-223, miR-16-1, miR-101-1, miR-124a-1, miR-26a-
1, miR-214, miR-27a, miR-24-1, miR-106a, miR-199a-1 and combinations thereof.
In yet another embodiment, the solid cancer is stomach cancer and the miR gene
product in the test sample is selected from the group consisting of miR-223,
miR-21,
miR-218-2, miR-103-2, miR-92-2, miR-25, miR-136, miR-191, miR-221, miR-125b-2,
miR-103-1, miR-214, miR-222, miR-212 prec, miR-125b-1, miR-100, miR-107, miR-
92-1, miR-96, miR-192, miR-23a, miR-215, miR-7-2, miR-138-2, miR-24-1, miR-
99b,
miR-33b, miR-24-2 and combinations thereof.
The level of the at least one miR gene product can be measured using a variety
of techniques that are well known to those of skill in the art (e.g.,
quantitative or semi-
6

CA 02811486 2013-03-26
=
quantitative RT-PCR, Northern blot analysis, solution hybridization
detection). In a
particular embodiment, the level of at least "one miR gene product is measured
by
reverse transcribing RNA from a test sample obtained from the subject to
provide a set
of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides
to one or
more miRNA-specific probe oligonucleotides (e.g., hybridzing to a microarray
that
comprises several miRNA-specific probe oligonucleotides) to provide a
hybrirli7ation
profile for the test sample, and comparing the test sample hybridization
profile to a
hybridization profile from a control sample. An alteration in the signal of at
least one
miRNA in the test sample relative to the control sample is indicative of the
subject
either having, or being at risk for developing, a solid cancer. In a
particular
embodiment, target oligonucleotides are hybridized to a microarray comprising
miRNA-specific.probe oligonucleotides for one or more miRNAs selected from the
group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,
miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-
107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and
combinations thereof.
The invention also encompasses methods of inhibiting tum.prigenesis in a
subject who has, or is suspected of having, a solid cancer (e.g., prostate
cancer, stomach
cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer), wherein
at least
one miR gene product is deregulated (e.g., down-regulated, up-regulated) in
the cancer
cells of the subject. When the at least one isolated miR gene product is down-
regulated
in the cancer cells, the method comprises administering an effective amount of
an
isolated miR gene product, an isolated variant or a biologically-active
fragment of the
miR gene product or variant, such that proliferation of cancer cells in the
subject is
inhibited. In a further embodiment, the at least one isolated miR gene product
is
selected from the group consisting of miR-145, miR-155, miR-218-2 and
combinations
thereof. In a particular embodiment, the miR gene product is not miR-15a or
miR-16-
1. When the at least one isolated miR gene product is up-regulated in the
cancer cells,
the method comprises administering to the subject an effective amount of at
least one
compound for inhibiting expression of the at least one miR gene product
(referred to
herein as a "miR expression-inhibition compound"), such that proliferation of
cancer
cells in the subject is inhibited. In a particular embodiment, the at least
one miR
7

CA 02811486 2013-03-26
expression-inhibition compound is specific for a miR gene product selected
from the
group consisting of miR-2I, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,
miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-
107, miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and
combinations thereof.
In a related embodiment, the methods of inhibiting tumorigenesis in a subject
additionally comprise the step of determining the amount of at least one miR
gene
product in cancer cells from the subject, and comparing that level of the miR
gene
product in the cells to the level of a corresponding miR gene product in
control cells. If
expression of the miR gene product is deregulated (e.g., down-regulated, up-
regulated)
in cancer cells, the methods further comprise altering the amount of the at
least one
miR gene product expressed in the cancer cells. In one embodiment, the amount
of the
miR gene product expressed in the cancer cells is less than the amount of the
miR gene
product expressed in a control cell (e.g., control cells), and an effective
amount of the
down-regulated miR gene product, isolated variant or biologically-active
fragment of
the miR gene product or variant, is administered to the subject. Suitable miR
gene
products for this embodiment include miR-I 45, miR-155, miR-2 18-2 and
combinations
thereof, among others. In a particular embodiment, the miR gene product is not
miR-
15a or miR-16-1. In another embodiment, the amount of the miR gene product
expressed in the cancer cells is greater than the amount of the miR gene
product
expressed in the control cell (e.g:, control cells), and an effective amount
of at least one
compound for inhibiting expression of the at least one up-regulated miR gene
product
is administered to the subject. Suitable compounds for inhibiting expression
of the at
least one miR gene product include, but are not limited to, compounds that
inhibit the
expression of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-
199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107,
miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations
thereof.
The invention further provides pharmaceutical compositions for treating solid
cancers (e.g., prostate cancer, stomach cancer, pancreatic cancer, lung
cancer, breast
cancer, colon cancer). In one embodiment, the pharmaceutical compositions
comprise
at least one isolated miR gene product and a pharmaceutically-acceptable
carrier. In a
8

CA 02811486 2013-03-26
particular embodiment, the at least one miR gene product corresponds to a miR
gene
product that has a decreased level of expression in cancer cells relative to
control cells.
In certain embodiments the isolated miR gene product is selected from the
group
consisting of miR-145, miR-155, miR-218-2 and combinations thereof.
In another embodiment, the pharmaceutical compositions of the invention
comprise at least one miR expression-inhibition compound and a
pharmaceutically-
acceptable carrier. In a particular embodiment, the at least one miR
expression-
inhibition compound is specific for a miR gene product whose expression is
greater in
cancer cells than in control cells. In certain embodiments, the miR expression-
inhibition compound is specific for one or more miR gene products selected
from the
group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,
miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-18 lb-1, miR-20a, miR-
107, miR-32, miR-214, miR-30c, miR-25, rniR-221, miR-106a and
combinations thereof.
The invention also encompasses methods of identifying an inhibitor of
tumorigenesis, comprising providing a test agent to a cell and measuring the
level of at
least one miR gene product in the cell. In one embodiment, the method
comprises
providing a test agent to a cell and measuring the level of at least one miR
gene product =
associated with decreased expression levels in solid cancers (e.g., prostate
cancer,
stomach cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer).
An
increase in the level of the miR gene product in the cell, relative to a
suitable control
cell, is indicative of the test agent being an inhibitor of tumorigenesis. In
a particular
embodiment, the at least one miR gene product associated with decreased
expression
levels in solid cancer cells is selected from the group consisting of miR-145,
miR-155,
miR-218-2 and combinations thereof.
In other embodiments, the method comprises providing a test agent to a cell
and
measuring the level of at least one miR gene product associated with increased
expression levels in solid cancers. A decrease in. the level of the miR gene
product in
the cell, relative to a suitable control cell, is indicative of the test agent
being an.
inhibitor of tumorigenesis. In a particular embodiment, the at least one miR
gene
product associated with increased expression levels in solid cancer cells is
selected
from the group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223,
miR-
9

CA 02811486 2013-03-26
128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a,
miR407,
miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations
thereof.
According to a further embodiment, there is provided a method of diagnosing
whether a
subject has, or is at risk for developing, a solid cancer, comprising:
measuring in a test sample
from the subject a level of miR-21 gene product and miR-191 gene product,
comparing the
level of miR-21 gene product and miR-191 gene product in the test sample to a
control level of
miR-21 gene product and miR-191 gene product; and diagnosing whether a subject
has, or is at
risk for developing, a solid cancer selected from the group consisting of
colon, lung, pancreas,
prostate and stomach cancer, wherein an increase in the level of miR-21 gene
product and miR-
191 gene product in the test sample, relative to the control level of a
corresponding miR-21
gene product and miR-191 gene product is indicative of the subject either
having, or being at
risk for developing, a solid cancer selected from the group consisting of:
colon, lung, pancreatic,
prostate and stomach cancer.
According to a still further embodiment, there is provided a method of
diagnosing
whether a subject has, or is at risk for developing, a solid cancer,
comprising: a. reverse
transcribing miR-21 RNA and miR-191 RNA from a test sample obtained from the
subject to
provide miR-21 and miR-191 oligodeoxynucleotides; b. hybridizing the miR-21
and miR-191
oligodeoxynucleotides to a microarray comprising miRNA-specific probe
oligonucleotides,
which includes miR-21 and miR-191 specific probe oligonucleotides, to provide
a hybridization
profile for the test sample; and c. comparing the test sample hybridization
profile to a control
hybridization profile, wherein, if a signal of miR-21 RNA and miR-191 RNA in
the test sample
hybridization profile is greater than the signal of miR-21 RNA and miR-191 RNA
in the control
hybridization profile, than the subject either has, or is at risk for
developing, a solid cancer
selected from the group consisting of colon, lung, pancreas, prostate and
stomach cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in
color. Copies of
this patent or patent application publication with color drawings will be
provided by the Office
upon request and payment of the necessary fee.
FIG. 1 depicts a clustering analysis of 540 samples, representing 6 solid
cancers (top)

CA 02811486 2013-03-26
and the respective nounal tissues. miRNAs included in the tree (n=137)
represent those whose
expression level (background-subtracted intensity) was higher than the
threshold value (256) in
at least 50% of the samples analyzed. Arrays were median-centered and
normalized using Gene
Cluster 2Ø Average linkage clustering was performed by using uncentered
correlation metric.
The shades indicate the difference in expression level from the median for the
microRNAs in
each sample.
FIG. 2 depicts unsupervised analysis of microRNA expression data. MicroRNA
profiling of 540 samples (indicated at top of panel) covering breast, colon,
lung, pancreas,
prostate and stomach (normal tissues and tumors) were filtered, centered and
normalized for
each feature. The data were subject to hierarchical clustering on both the
samples (horizontally-
oriented) and the features (vertically-oriented) with average linkage and
Pearson correlation as
similarity measure. Sample names are indicated at the top of the figure and
miRNA names on
the left. The probe ED is indicated in parentheses, as the same microRNA can
be measured by
different oligonucleotides.
FIG. 3 depicts the expression of differentially-regulated miRNAS across solid
cancers
(top). Sixty-one microRNAs, which are present in at least 90% of the tissues
solid cancers, are
represented (right of panel). The tree displays the average absolute
expression values for each of
the listed microRNAs after log2transformation. The mean was computed over all
samples from
the same tissue or tumor histotype. Genes were mean-centered and normalized
using Gene
Cluster 2Ø Average linkage clustering was performed using Euclidean
distance.
10a

CA 02811486 2013-03-26
FIG. 4 depicts fold changes in the expression of miRNAs present in at least
75% of the solid tumors with at least 1 tumor absolute value higher than 2 in
different
cancer samples (top), relative to normal samples. The tree displays the log2
transformation of average fold changes (cancer vs. normal). The mean was
computed
over all samples from the same tissue or tumor histotype. Arrays were mean-
centered
and normalized using Gene. Cluster 2Ø Average linkage clustering was
performed
using uncentered correlation metric.
FIG. 5 depicts fold changes in the expression of miRNAs present in the
signatures of at least 50% of the solid tumors in cancer vs. normal samples.
The tree
displays the log 2 transformation of the average fold changes (cancer over
normal). The
mean was computed over all samples from the same tissue or tumor histotype.
Arrays
were mean centered and normalized using Gene Cluster 2Ø Average linkage
clustering
was performed using uncentered correlation metric.
FIG. 6A depicts bar graphs indicating that the 3 'UTR of different genes
encoding cancer protein enables cancer regulation by microRNA. The relative
repression of firefly luciferase expression (Fold Change) standardized to a
renilla
luciferase control. PLAG1, pleiomorphic adenoma gene 1; TGFBR2, transforming
growth factor beta receptor II; Rb, retinoblastoma gene. pGL-3 (Promega) was
used as
the empty vector. miR-20a, miR-26a-1 and riziR-106 oligoRNAs (sense and
scrambled)
were used for transfections. A second experiment using mutated versions of
each target
'1112NA, which lack the 5' tniRNA-end complementarity site (MUT), as controls
is
shown in the bottom panel. All the experiments were performed twice in.
triplicate
(n=6).
= FIG. 6B depicts Western blots indicating that, in certain cancers (e.g.,
lung,
breast; colon, gastric), the levels of RB1 (Rb) protein displays an inverse
correlation
with the level of miR-106a expression. 13-Actin was used as a control for
normalization. Ni, normal sample; Ti and T2, tumor sample.
FIG. 7 depicts Northern blots showing down-regulation of miR-145 (top) and
up-regulation of miR-21 (bottom) expression in breast cancer samples (P series
and
numbered series) relative to normal samples. Normalization was performed with
a U6-
specific probe.
11
=

CA 02811486 2013-03-26
FIG. 8 depicts Northern blots showing up-regulation of miR-103 and down-
regulation miR-155 (top) expression in different endocrine pancreatic cancer
samples
(WDET, well differentiated pancreatic endocrine tumors, WDEC, well
differentiated
pancreatic endocrine carcinomas and ACC, pancreatic acinar cell carcinomas)
relative
to normal samples (K series), as well as up-regulation of miR-204 (bottom)
expression
in insulinomas (F series) relative to normal samples (K series) and non
secreting/non
functioning (NF-series) samples. Normalization was performed with a probe
specific to
58 RNA.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based, in part, on the identification of particular
microRNAs whose expression is altered in cancer cells associated with
different solid
cancers, such as colon, stomach, pancreatic, lung, breast and prostate cancer,
relative to
normal control cells.
As used herein interchangeably, a "miR gene product," "microRNA," "miR," or
= "miRNA" refers to the unprocessed (e.g., precursor) or processed (e.g.,
mature) RNA
transcript from a miR gene. As the miR gene products are not translated into
protein,
the term "miR gene products" does not include proteins. The unprocessed miR
gene
transcript is also called a "miR precursor" or "miR prec" and typically
comprises an
RNA transcript of about 70-100 nucleotides in length. The miR precursor can be
processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse
III
(e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNA molecule. This
active
19-25 nucleotide RNA molecule is also called the "processed" miR gene
transcript or
"mature" miRNA.
The active 19-25 nucleotide RNA molecule can be obtained from the miR
precursor through natural processing routes (e.g., using intact cells or cell
lysates) or by
synthetic processing routes (e.g., using isolated processing enzymes, such as
isolated
Dicer, Argonaut, or RNAse III). It is understood that the active 19-25
nucleotide RNA
molecule can also be produced directly by biological or chemical synthesis,
without
having been processed from the miR precursor. When a microRNA is referred to
herein by name, the name corresponds to both the precursor and mature forms,
unless
otherwise indicated.
12.

CA 02811486 2013-03-26
Tables la and lb depict the nucleotide sequences of particular precursor and
mature human microRNAs.
Table la- Human microRNA Precursor Sequences
Precursor Sequence (5' To 31)* SEQ 1D
Name NO.
let-7a-1 CACUGUGGGAUGAGGUAGUAGGUUGUAUAGUUU
UAGGGUCACACCCACCACUGGGAGAUAACUAUA 1
CAAUCUACUGUCUUUCCUAACGUG
let-7a-2 AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUU
ACAUCAAGGGAGAUAACUGUACAGCCUCCUAGC 2
UUUCCU
let-7a-3 GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUC
UGCCCUGCUAUGGGAUAACUALTACAAUCUACUG 3
UCUUUCCU
let-7a-4 GUGACUGCAUGCUCCCAGGUUGAGGUAGUAGGU
UGUAUAGUUUAGAAUUACACAAGGGAGAUAACU 4
GUACAGCCUCCUAGCUUUCCUUGGGUCUUGCAC -
UAAACAAC
let-7b GGCGGGGUGAGGUAGUAGG'UUGUGUGGUUUCAG
GGCAGUGAUGUUGCCCCUCGGAAGAUAACUAUA
CAACCUACUGCCUUCCCUG
let-7c GCAUCCGGGUUGAGGUAGUAGGUUGUAUGGUUU
AGAGUUACACCCUGGGAGLTUAACUGUACAACCU 6
UCUAGCUUUCCUUGGAGC
let-7d CCUAGGAAGAGGUAGUAGGUUGCAUAGUUTJUAG
GGCAGGGAUUUUGCCCACAAGGAGGUAACUAUA 7
CGACCUGCUGCCUUUCUUAGG
let-7d-v1 CUAGGAAGAGGUAGUAGUUUGCAUAGUUUUAGG
GCAAAGAUUUUGCCCACAAGUAGUUAGCUAUAC
GACCUGCAGCCUUUUGUAG
let-7d-v2 CUGGCUGAGGUAGUAGUUTJGUGCUGUUGGUCGG
GUUGUGACAUUGCCCGCUGUGGAGAUAACUGCG 9
CAAGCUACUGCCUUGCUAG
let-7e CCCGGGCUGAGGUAGGAGGIJUGUAUAGUUGAGG
AGGACACCCAAGGAGAUCACUAUACGGCCUCCU 10
AGCUUUCCCCAGG
let-7f-1 UCAGAGUGAGGUAGUAGA1UUGUAUAGUUGUGGG
GUAGUGAUUUTJACCCUGUUCAGGAGAUAACUAU 11
ACAAUCUAUUGCCUUCCCUGA
let-7f-2-1 CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUGUG
GGGUAGUGAUUUUACCCUGUUCAGGAGAUAACU 12
AUACAAUCUALTUGCCUUCCCUGA
13 =

CA 02811486 2013-03-26
Precursor Sequence (5' To 31)* SEQ ED
Name NO.
let-7f-2-2 CUGUGGGAUGAGGUAGUAGAUUGUAUAGUIJUUA "
GGGUCAUACCCCAUCUUGGAGAUAACUAUACAG 13
=
UCUACUGUCUUUCCCACGG
let-7g UUGCCUGAUUCCAGGCUGAGGUAGUAGUUUGUA
CAGUUUGAGGGUCUAUGAUACCACCCGGUACAG 14
GAGAUAACUGUACAGGCCACUGCCUUGCCAGGA
ACAGCGCGC
let-7i CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGG
GUUGUGACAUUGCCCGCUGUGGAGAUAACUGCG 15
CAAGCUACTJGCCUUGCUAG
rrziR-1b-1-1 ACCUACUCAGAGUACAUACUUCUUUAUGUACCC
AUAUGAACAUAC.AAUGCUAUGGAAUGUAAAGAA 16
GUAUGUATJUUUUGGUAGGC
miR-lb-1-2 CAGCUAACAACUUAGUAAUACCUACUCAGAGUA
CAUACUUCUUUAUGUACCCAUAUGAACAUACAA 17
UGCUAUGGAAUGUAAAGAAGUAUGUAUUUUUGG
UAGGCAAUA
miR-1 b-2 GCCIJGCUUGGGAAACAUACUUCLTUUAUAUGCCC
AUAUGGACCUGCUAAGCUAUGGAAUGUAAAGAA 18
GUAUGUAUCUCAGGCCGGG
miR-1 b UGGGAAACAUACUUCUUUAUAUGCCCAUAUGGA
CCUGCUAAGCUAUGGAAUGUAAAGAAGUAUGTJA 19
UCUCA
miR-1d ACCUACUCAGAGUACAUACUUCUUUAUGUACCC
AUAUGAACAUACAAUGCUAUGGAAUGUAAAGAA 20
GUAUGUAUUUUUGGUAGGC
zrziR-7-1 a TJGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAG
UGAUUUTJGUUGLTUUUUAGAUAACUAAAUc GACA 21
ACAAATICACAGUCUGCCAUAUGGCACAGGCCAU
GCCUCUACA
miR-7-1 b UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUA
GUGATJULTUGUUGUUUTTUAGAUAACUAAAUCGAC 22
AACAAAUCACAGUCUGCCAUAUGGCACAGGCCA
UGCCUCUACAG
miR-7-2 CUGGAUACAGAGUGG-ACCGGCUGGCCCCAUCUG
GAAGACUAGUGAULTUUGUUGUUGUCUUACUGCG 23
CUCAACAACAAAUCCCAGUCUACCUAAUGGUGC
CAGCCAUCGCA
miR-7-3 AGAUUAGAGUGGCUGUGGUCUAGUGCTJGUGUGG
AAGACUAGUGAUUUUGUUGUUCUGAUGUACUAC 24
GACAACAAGUCACAGCCGGCCUCAUAGCGCAGA
CUCCCUUCGAC
rniR-9-1 CGGGGUUGGUUGUUAUCUUUGGUUAUCUAGCUG
UAUGAGUGGUGUGGAGUCUUCAUAAAGCUAGAU 25
AACCGAAAGUAAAAAUAACCCCA
14
=

CA 02811486 2013-03-26
=
Precursor sequence (5' To 31)*
SEQ ID
Name NO.
miR-9-2 GGAAGCGAGUUGUUAUCUUUGGUUAUCUAGCUG
UAUGAGUGTJAUUGGUCUUCAUAAAGCUAGAUAA 26
CCGAAAGUAAAAACUCCUUCA
miR-9-3 GGAGGCCCGUUUCTJCUCUUUGGIJUAUCUAGCUG
UAUGAGUGCCACAGAGCCGUCAUAAAGCLTAGAU 27
AACCGAAAGUAGAAAUGAUUCUCA
miR-1 Oa GAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAU
CCGAAUUUGUGUAAGGAAUUUUGUGGUCACAAA 28
UUCGUAUCUAGGGGAAUAUGUAGUUGACAUAAA
CACUCCGCUCU
miR-10b CCAGAGGUUGTJAACGUUGUCUAUAUAUACCCUG
UAGAACCGAAUUUGUGUGGUAUCCGUAUAGUCA 29
CAGAUUCGAL.JUCUAGGGGAAUAUAUGGUCGAUG
CAAAAACUUCA
miR-15a-2 GCGCGAAUGUGUGUUTJAAAAA.AAAUAAAA.CCUU
GGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGA 30.
UUUUGAAAAGGUGCAGGCCAUAUUGUGCUGCCU
CAAAAAUAC
miR- 15a CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUG
UGGAUUUUGAAAAGGUGCAGGCCAUAUUGUGCU 31
GCCUCAAAAAUACAAGG
miR-15b-1 CTJGUAGCAGCACAUCAUGGUUUACAUGCUACAG
L.UCAAGAUGCGAAUCAUUAUUUGCLJGCUCUAG 32
miR-15b-2 UUGAGGCCUUAAAGUACUGUAGCAGCACAUCATJ_
GGULTUACAUGCUACAGUCAAGATJGCGAAUCAUU 33
AUUUGCUGCUCUAGAAAUUUAAGGAAAUUCAU
miR-16-1 GUCAGCAGUGCCUUAGCAGCACGUAAAUAUTIGG
CGULJAAGAUUCUAAAAUUAUCUCCAGUAUUAAC 34
UGUGCUGCUGAAGUAAGGUUGAC
miR-1 6-2 OLTUCCACUCUAGCAGCACGUAAATJAUUGGCGUA
GUG-AAATJAUAUAUUAAACACCAAUAUUACUGUG 35
CUGCUUUAGUGUGAC
miR-16-13 GCAGUGCCUUAGCAGCACGUAAAUAUUGGCG_UU
AAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUG 36
CUGCUGAAGUAAGGU
miR-17 GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGG '
UAGUGAUAUGUGCAUCUACUGCAGUGAAGGCAC 37
UUGUAGCAUUAUGGUGAC
miR-18 UGULICUAAGGUGCAUCUAGUGCAGAUAGUGAAG
= TJAGAUUAGCAUCUACUGCCCUAAGUGCUCCUUC 38
UGGCA
miR-18-13 UUUUUGUUCUAAGGUGCAUCUAGUGCAGAUAGU
= GAAGUAGAUUAGCAUCUACUGCCCUAAGUGCUC 39
CLTUCUGGCAUAAGAA =

CA 02811486 2013-03-26
Precursor Sequence (5 To 31)*
SEQ ED
Name
NO.
miR-19a GCAGUCCUCUGUUAGOULTUGCAUAGUUGCACUA
CAAGAAGAAUGUAGUUGUGCAAAUCUAUGCAAA 40
ACUGAUGGUGGCCUGC
=
miR-19a-13 CAGUCCUCUGLTUAGUUUUGCAUAGUUGCACUAC
AAGAAGAAUGUAGUUGUGCAAAUCUAUGCAAAA 41
CUGAUGGUGGCCUG
miR-19b-1 CACUGLTUCUAUGGUUAGULJUUGCAGGUUUGCAU
CCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAU 42
GCAAAACUGACUGUGGUAGUG
miR-19b-2 ACATJUGCUACUUACAAUUAGUUtNJGCAGGULTUG
CAUUUCAGCOUAUAUAUGUAUAUGUGGCUGUGC 43
AAAUCCAUGCAAAACUGAUUGUGAUAAUGU
miR-19b-13 UUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGC
UGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAA 44
ACUGACUGUGGUAG
miR-19b-X UUACAAUUAGLTUTIUGCAGGUUUGCAUUUCAGCG
UAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGC 45
AAAACUGAUUGUGAU
miR-20 GUAGCACUAAAGUGCUUAUAGUGCAGGUAGUGU
(miR-20a) UUAGLTUAUCUACUGCALTUAUGAGCACUUAAAGU 46
ACUGC
miR-21 LIGUCGGGUAGCUTJAUCAGACUGAUGUUGACUGU
UGAAUCUCAUGGCAACACCAGUCGAUGGGCUGTJ 47
CUGACA
mIR-21-17 ACCUUGUCGGGUAGCUUAUCAGACUGAUGUUGA
CUG-TJUGAAUCUCAUGGCAACACCAGUCGAUGGG 48
CUGUCUGACAUULTUG
miR-22 GGCUGAGCCGCAGUAGUUCUUCAGUGGCAAGCU
UUAUGUCCUGACCCAGCUAAAGCUGCCAGUUGA 49
AGAACUGIJUGCCCUCITGCC
miR-23a GGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCU
UCCUGUCACAAAUCACAUUGCCAGGGAUUUCCA 50
ACCGACC
miR-23b CUCA.GGUGCUCUGGCUGCUUGGGUUCCUGGCAU
GCUGAUUUGUGACUUAAGAITUAAAAUCACAUUG 51
CCAGGGAUUACCACGCAACCACGACCUUGGC
miR:23-19 CCACGGCCGGCUGGGGUUCCUGGGGAUGGGAUTJ
UGCUUCCUGUCACAAAUCACAUUGCCAGGGALTU 52
UCCAACCGACCCUGA
miR-24-1 CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCA
UUUUACACACUGGCUCAGUUCAGCAGGAACAGG 53
AG
miR-24-2 -CUCUGCCUCCCGUGCCUACUGAGCUGAAACACAG
UUGGUUUGUGUACACUGGCUCAGUUCAGCAGGA 54
ACAGGG
16

CA 02811486 2013-03-26
=
Precursor Sequence (5' To 3')* SEQ ID
Name - NO.
rniR-24-19 CCCUGGGCUCUGCCUCCCGUGCCUACUGAGCUGA
AACACAGUUGGUUUGUGUACACUGGCUCAGUUC 55
AGCAGGAACAGGGG
miR-24-9 CCCUCCGGUGCCUACUGAGCUGAUAUCAGUUCU
CAUUUUACACACUGGCUCAGUUCAGCAGGAACA 56
GCAUC
miR-25 GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUU
GCUGGACGCUGCCCUGGGCAUUGCACUUGUCUC 57
GGUCUGACAGUGCCGGCC
miR-26a AGGCCGUGGCCUCGUUCAAGUAAUCCAGGAUAG
GCUGUGCAGGUCCCAAUGGCCUAUCUUGGUUAC 58
UUGCACGGGGACGCGGGCCU
miR-26a-1 GUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGil
GCAGGUCCCAAUGGGCCUALTUCUUGGUUACUUG 59
CACGGGGACGC
miR-26a-2 GGCUGUGGCUGGAUUCAAGUAAUCCAGGAUAGG
CUGUUUCCAUCUGUGAGGCCUAUUCUUGAUUAC 60
UUGULTUCUGGAGGCAGCU
miR-26b CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUU
GUGUGCUGUCCAGCCUGUUCUCCAUUACUUGGC 61
UCGGGGACCGG
mil?-27a CUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGG
GUCCACACCAAGUCGUGUUCACAGUGGCUAAGU_ 62
UCCGCCCCCCAG
miR-27b-1 AGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGA
UUGGUUUCCGCUUUGUUCACAGUGGCUAAGUUC 63
UGCACCU
miR-27b-2 ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUU
GGUGAACAGUGAUUGGUUUCCGCUUUGUUCACA 64
GUGGCUAAGUUCUGCACCUGAAGAGAAGGUG
miR-27-19 . CCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAG
GGUCCACACCAAGUCGUGUUCACAGUGGCUAAG: 65
UUCCGCCCCCCAGG
miR-28 GGUCCUUGCCCUCAAGGAGCUCACAGUCUAUUG
AGUUACCUUUCUGACUUUCCCACUAGAUUGUGA. 66
GCUCCUGGAGGGCAGGCACU
miR-29a-2 CCUUCUGUGACCCCUUAGAGGAUGACUGAUUUC
UUUUGGUGUUCAGAGUCAAUATJAAUUUUCUAGC 67
ACCAUCUGAAAUCGGUUAUAAUGAUUGGGGAAG
AGCACCAUG
miR-29a AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUA
UAAUUUUCUAGCACCAUCUGAAAUCGGUUAU 68
miR-29b-1 CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGA
UUUAAAUAGUGAUUGUCUAGCACCAUUUGAAAU 69
CAGUGUUCUUGGGGG
. 17

CA 02811486 2013-03-26
Precursor Sequence (5' To 31)* SEQ ED
Name NO.
miR-29b-2 CUUCUGGAAGCUGGUUUCACALTGGUGGCUUAGA
UUUUUCCAUCLTUUGUAUCUAGCACCAUUUGAAA 70
UCAGUGUUUUAGGAG
miR-29c ACCACUGGCCCAUCUCUUACACAGGCUGACCGAU
UUCUCCUGGUGUUCAGAGUCUGUVUUUGUCUAG 71
CACCAUUUGAAAUCGGUUAUGAUGUAGGGGGAA
AAGCAGCAQC
miR-30a GCGACUGUAAACAUCCUCGACUGGAAGCUGUGA
AGCCACAGAUGGGCUUUCAGUCGGAUGUUUGCA 72
GCUGC
miR-30b-1 AUGUAAACAUCCUACACUCAGCUGUAAUACAUG
GAUUGGCUGGGAGGUGGAUGUUUACGU 73
miR-30b-2 ACCAAGUUUCAGUUCAUGUAAACAUCCUACACU
AGCUGUAAUACAUGGAUUGGCUGGGAGGUGGA 74
UGUUUACUUCAGCUGACUUGGA
miR-30c AGAUACUGUAAACAUCCUACACUCUCAGCUGUG
GAAAGUAAGAAAGCUGGGAGAAGGCUGUUUACU 75
C'UUUCU =
miR-30d GUUGLTUGUAAACAUCCCCGACUGGAAGCUGUAA
GACACAGCUAAGCUUUCAGUCAGAUGUIJUGCUG 76
CUAC
miR-30e CUGUAAACAUCCUUGACLTGGAAGCUGUAAGGUG
UUCAGAGGAGCUUUCAGUCGGAUGUUUACAG 77
miR-31 GGAGAGGAGGCAAGAUGCUGGCAUAGCUGUUGA
ACUGGGAACCUGCUAUGCCAACAUALTUGCCAUC 78
UUUCC
miR-32 GGAGAUAUUGCACAUUACUAAGUUGCAUGLTUGU
CACGGCCUCAAUGCAAUUUAGTJGUGUGUGAUAU 79
UULIC
miR-33b GGGGGCCGAGAGAGGCGGGCGGCCCCGCGGUGC
AUUGCUGUUGCAUUGCACGUGUGUGAGGCGGGU 80
GCAGUGCCUCGGCAGUGCAGCCCGGAGCCGGCCC
CUGGCACCAC
miR-33b-2 ACCAAGUUUCAGUUCAUG-UAAACAUCCUACACU
CAGCUGUAAUACAUGGAUUGGCUGGGAGGUGGA 81
UGUULTACUUCAGCUGACUUGGA
miR-33 CUOUGGUGCAUUGUAGUUGCAUUGCAUGUUCUG
GUGGUACCCAUGCAAUGUUUCCACAGUGCA UCA 82
CAG
miR-34-a GGCCAGCUGUGAGUGUUUCU'UUGGCAGUGUCUU
AG CUGG UUGUUGUGAGCAAUAGIJAA GGAAGC AA 83
UCAGCAAGUAUACUGCCCUAGAAGUGCUGCACG
UUGUGGGGCCC
18

CA 02811486 2013-03-26
=
Precursor Sequence (5' To 3')* SEQ ID
Name NO.
miR-34-b GUGCUCGGUUUGUAGGCAGUGUCALTUAGCUGAU
UGUACUGUGGUGGUUACAAUCACUAACUCCACU 84
GCCAUCAA.AACAAG-GCAC
' miR-34-c AGUCUAGLTUACUAGGCAGUGUAGUUAGCUGAUU
GCUAAUAGUACCAAUCACU.AACCACACGGCCAG 85
GUAAAAAGAUU =
miR-9 1-13 UCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGU
AGUGAUAUGUGCAUaUACUGCAGUGAAGGCACU 86
UGUAGCAUUAUGGUGA
miR-92-1 CUUUCUACACAGGCTUGGGAUCGGUUGCAAUGCU
GUGUUUCUGUAUGGUAUUGCACUUGUCCCGGCC 87
UGUUGAGUUUGG
miR-92 -2 UCAUCCCUGGGUGGGGAUUUGLTUGCAUUACUUG
UGUUCUAUAUAAAGUALTUGCACUUGUCCCGGCC 88.
UGU_GGAAGA
miR-93-1 CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAG
(miR-93-2) UGUGALTUACCCAACCUACUGCUGAGCUAGCACU 89
UCCCGAGCCCCCGG
miR-95-4 AACACAGUGGGCACUCAAUAAAUGUCUGUUGAA
UUGAAAUGCGUUACAUUCAACGGGUAUUUAUUG 90
AGCACCCACUCUGUG
miR-96-7 UGGCCGAUUUUGGCACUAGCACAUULTUUGCUUG =
UGUCUCUCCGCUCUGAGCAAUCAUGUGCAGUGC 91
CAAUAUGGGAAA
rniR-97-6 GUGAGCGACUGUAAACAUCCUCGACUGGAAGCU .
(m1R-30*) . GUGAAGCCACAGAUGGGCUUUCAGUCGGAUGUU 92
UGCAGCUGCCUACU
miR-98 GLTGAGGUAGUAAGUUGUAUUGLTUGUGGGGUAGG
GAUALTUAGGCCCCAAUUAGAAGAUAACUAUACA 93
ACIJUACUACUUTJCC
miR-99b GGCACCCACCCGUAGAACCGACCUUGCGGGGCCU
UCGCCGCACACAAGCUCGUGUCUGUGGGUCC GU 94
GUC
miR-99a CCCAUUGGCAUAAACCCGUAGAUCCGAUCUUGU
GGUGAAGUGGACCGCACAAGCUCGCUUCUAUGG 95
GUCUGUGUCAGUGUG
miR-100-1/2 AAGAGAGAAGAUAUUGAGGCCUGUUGCCACAAA
CCCGUAGAUCCGAACUUGUGGUAUUAGUCCGCA 96
CAAGCUUGUAUCUAUAGGUAUGUGUCUGUUAGG
CAATJCUCAC
miR-100-11 CCUGUUGCCACAAACCCGUAGAUCCGAAC UUGU
GGUAUUAGUCCGCACAAGCUUGUAUCUAUAGGU 97
AUGUGUCUGUUAGG
19

CA 02811486 2013-03-26
=
Precursor - Sequence (5' To 3')*
SEQ
Name
NO.
miR-101 -1 /2 = A GGCUGCCCUGGCUCAGUUAUCACAGUGCUGAU
= GCUGUCUAULTCUAAAGGUACAGUACUGUGAUAA 98
=
CUGAAGGAUGGCAGCCAUCUUACCUUCCAUCAG
AGGAGCCUCAC
miR-101 UCAGUUAUCACAGUGCUGAUGCUGUCCAUUCUA
AAGGUACAGUACUGUGAUAACUGA 99
miR-101-1 UGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUG
UCUAUUCU.AAAGGUACAGUACUGUGAUAACUGA 100
AG GAUGGCA
miR-101-2 ACUGUCCUULTUUCGGUUAUCAUGGUACCGAUGC
UGUAUAUCUG.AAAGGUACAGUACUGUGAUAA CU 101
GAAGAAUGGUGGU
miR-101 -9 TJGUCCUUUUUCGGUUAUCAUGGUACCGAUGCUG
UAUAUCUG.AAAGGUACAGUACUGUGAUAACUGA 102
AGAAUGGUG
miR-102-1 CLTUCUGGAAGCUGGUUUCACAUGGUGGCUUAGA .
UUUUUCCAUCUUUGUAUCUAGCACCALTUUGAAA. 103
UCAGUGUUUUAGGAG
miR-102-7. 1 CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGA
(mi.R-1 02- UUUAAAUAGUGAUUGUCUAGCACCAUUUGAAAU 104
7.2) CAGUGUUCUUGGGGG
miR-103-2 LTUGUGCUUUCAGCUUCUUUACAGUGCUGCCUUG
UAGCAUUCAGGUCAAGCAACAUUGUACAGGGCU = 105
AUGAAAGAACCA
miR-103 -1 UACUGCCCUCGGCLTUCUUUACAGUGCUGCCUUG
UUGCAUAUGGAUCAAGCAGCAUUGUACAGGGCU 106
AUGAAGGCALTUG
miR-104-17 AAAUGUCAGACAGCCCAUCGACUGGUGUUGCCA
UGAGALTUCAACAGUCAACAUCAGUCUGAUAA.GC 107
LIACCCGACAAGG
miR-1 05-1 UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUG
GUGGCUGCUCAUGCACCACGGAUGUUUGAGCAU 108
GLIGCUACGGUGUCUA
miR-105-2 UGUGCAUCGUG GUCAAATJGCUCAGACUCCUGUG
GUGGCU GCLTUAUG CAC CAC GGAUGUUUGAGCAU .109
GIJGCUAUGGUGUCUA
miR-10 6-a CCUUGGCCAUGUAAAAGUGCUUACAGUGCAGGU
AGCUUUUUGAGAUCUACUGCAAUGUAAGCACUU 110
CUUACAUUACCAUGG
miR-106-b CCUGCCGGGGCUAAAGUGCUGACAGUGCAGAUA
GUGGUCCUCUCCGUGCUACCGCACUGUGGGUAC 111
ULTGCUGCUCCAGCAGG
= miR-107 CUCUCUGCUUUCAGCUUCUUUACAGUGUUGCCU
UGUGGCAUGGAGUUCAAGCAGCAUUGUACAGGG 112
CUAUCAAAGCACAGA

CA 02811486 2013-03-26
Precursor Sequence (5' To 35* SEQ ID
Name NO.
miR-108-1- ACACUGCAAGAACAAUAAGGAUUULTUAGGGGCA
small UUAUGACUGAGUCAGAAAACACAGCUGCCCCUG 113
AAAGUCCCUCAUUUUUCUUGCUGU
mi.R-108-2- ACUGCAAGAGCANUAAGGAUUUUUAGGGGCAUU
small AUGAUAGUGGAAUGGAAACACAUCUGCCCCCAA 114
AAGUCCCUCAUUUU
miR-122a-1 CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGU
LTUGUGUCUAAACUAUCAAAC6CCAUUAUCACAC 115
UAAAUAGCUACUGCUAGGC
miR-122a-2 AGCUGUGGAGUGUGACAAUGGUGUUUGUGUCCA
AACUAUCAAACGCCAUUAUCACACUAAAUAGCU 116
miR-123 ACALTUAUUACUUUUGGUACGCGCUGUGACACUU
CAAACUCGUACCGUOAGUAAUAAUGCGC 117
miR-124a-1 AGGCCUCUCUCUCCGUGUUCACAGCGGACCUUG
AUUUAAAUGUCCAUACAAUUAAGGCACGCGGUG 118
AAUGCCAAGAAUGGGGCUG
miR-124a-2 AUCAAGAUUAGAGGCUCUGCUCUCCGUGUUCAC
AGCGGACCUUGAUUUAAUGUCAUACAAUUAAGG 119
CACGCGGUGAAUGCCAAGAGCGGAGCCUACGGC
UGCACUUGAAG
miR-124a-3 UGAGGGCCCCUCUGCGUGUUCACAGCGGACCUU
GAUUUAAUGUCUAUACAAUUAAGGCACGCGGUG 120
AAUGCCAAGAGAGGCGCCUCC
miR-124a CUCUGCGUGUUCACAGCGGACCUUGAULTUAAUG
UCUAUACAAUUAAGGCACGCGGUGAAUGCCAAG 121
AG
miR-124b CUCUCCGUGUUCACAGCGGACCUUGAUUUAAUG
UCAUACAAUUAAGGCACGCGGUGAAUGCCAAGA 122
miR-12.5a-1 UGCCAGUCUCUAGGUCCCUGAGACCCUUUAACC
UGUGAGGACAUCCAGGGUCACAGGUGAGGUUCU 123
UGGGAGCCUGGCGUCUGGCC
miR-12 5a-2 GGUCCCUGAGACCCUUUAACCUGUGAGGACAUC
CAGGGUCACAGGUGAGGUUCUUGGGAGCCUGG 124
miR-125b-1 UGCGCUCCUCUCAGUCCCUGAGACCCUAACUUGU
GAUGUUUACCGUUUAAAUCCACGGGUUAGGCUC 125
UUGGGAGCUGCGAGUCGUGCU
rniR-125b-2 ACCAGACUUUUCCUAGUCCCUGAGACCCUAACU
UGUGAGGUAUUUUAGUAACAUCACAAGUCAGGC 126
UCUUGGGAtCUAGGCGGAGGGeA
miR-126-1 CGCUGGCGACGGGACAUUAUUACUUUUGGUACG
CGCUGUGACACUUCAAACUCGUACCGUGAGUAA 127
UAAUGCGCCGUCCACGGCA
miR-126-2 ACAUUAUUACUUUUGGUACGCGCUGUGACACUU
CAAACUCGUA.CCGUGAGUAA.UAAUGCGC 128
21

CA 02811486 2013-03-26
Precursor Sequence (5' To 31)* SEQ ID
Name NO.
= miR-127-1 UGUGAUCACUGUCUCCAGCCUGCUGAAGCUCAG
AGGGCUCUGAUUCAGAAAGAUCAUCGGAUCCGU 129
CUGAGCLTUGGCUGGUCGGAAGUCUCAUCAUC
miR-127-2 CCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCA
GAAAGAUCAUCGGAUCCGUCUGAGCUUGGCUGG 130
UCGG
miR-128a UGAGCUGUUGGAUUCGGGGCCOUAGCACUGUCU
GAGAGGUUUACAUUUCUCACAGUGAACCGGUCU 131
CUUUUUCAGCUGCUUC
miR-128b GCCCGGCAOCCACUGLJGCAGUGGGAAGGOGGGC
CGAUACACUGUACGAGAGUGAGUAGCAGGTJCUC 132
ACAGUGAACCGGUCUCUUUCCCUACUGUGUCAC
ACUdCUAAUGG
mi.R-128 GUUGGALTUCGGGGCCGUAGCACUGUCUGAGAGG
UUUACAUUUCUCACAGUGAACCGGUCUCUUTJTJU 133
CAGC
miR-129-1 UGGAUCUUUUUGCGGUCUGGGCUUGCUGUUCCU
CUCAACAGUAGUCAGGAAGCCCUUACCCCAAAA 134
AGUAUCUA
miR-129-2 UGCCCUUCGCGAAUCUUUUUGCGGUCUGGGCUU
GCUGUACAUAACUCAAUAGCCGGAAGCCCLTUAC 135
CCCAAAAAGCAULTUGCGGAGGGCG
miR-130a UGCUGCUGGCCAGAGCUCUUUUCACAUUGUGCU
ACUGUCUGCACCUGUCACUAGCAGUGCAAUGUU 136
AAAAGGGCAUUGGCCGUGUAGUG
miR-131 -1 Gc CAGGAGGCGGGGUUGGUUGUUAUCUUUGGUU
AUCUAGCUGUAUGAGUGGUGUGGAGUCUUCAUA 137
AAGCUAGAUAACCGAAAGUAAAAAUAACCCCAU
ACACTJGCGCAG
-
miR-131 -3 CAC G G C GC G GCAG C GG CA C UGGCUAAGGGAGGC
CCGUUUCUCUCUBUGGUUAUCUAGCUGUAUGAG 1 3S
UGCCACAGAGCC GUCAUAAAGCUAGAUAACCGA
AAGUAGAAAUG
miR-131 GUUGUUAUCUUUGGLTUAUCUAGCTJGUAUGAGUG
UAUUGGUCUUCAUAAAGCUAGAUAACCGAAAGU 139
AAAAAC
rniR-132-1 CCGCCCCCGCGUCUCCAGGGCAACCGUGGCUUUC
GALTUGUUACUGUGGGAACUGGAGGUAACAGUCU 140
ACAGCCAUGGUCGCCCCGCAGCACGCCCACGCGC
miR-132-2 GGGCAACCGUGGCUUUCGAUUGUUACUGUGGGA
ACUGGAGGUAACAGUC1UACAGCCAUGGUCGCCC 141
miR-133 a-1 ACAAUGCUUUGCUAGAGCUGGUAAAAUGGAACC
AAAUCGCCUCUUCAAUGGAUTJUGGUCCCCUUCA 142
ACCAGCUGUAGCUAUGCAUUGA
22

CA 02811486 2013-03-26
Precursor Sequence (5' To 31)* SEQ ID
Name NO.
miR-133a-2 GGGAGCCAAAUGCUUUGCUAGAGCUGGUAAAA.0
GGAACCAAAUCGACUGUCCAAUGGAUUUGGUCC 143
CCLTUCAACCAGCUGUAGCTJGUGCAUUGAUGGCG
CCG
miR-133 GCUAGAGCUGGUAAAAUGGAACCAAAUCGCCUC
UUCAAUGGAUTJUGGUCCCCUEJCAACCAGCUGUA 144
GC
miR-133b CCUCAGAAGAAAGAUGCCCCCUGCUCUGGCUGG
=
TJCAAACGGAACCAAGUCCGUCUUCCUGAGAGGU 145
UUGGUCCCCUUCAACCAGCUACAGCAGGGCUGG
CAAUOCCCAGUCCUUGGAGA
miR-133b- GCCCCCUGCUCUGGCUGOUCAAACGGAACCAAG
small UCCGUCUUCC UGAGAGGUTJUGGUCCCCUUCAAC 146
CAGCUACAGCAGGG
miR-134-1 CAGGGUGUGUGACUGGUUGACCAGAGGGGCAUG -
CACUGUGUUCACCaUGUGGGCCACCLTAGUCACCA 147
ACCCUC
miR-134-2 AGGGTJGUGUGACUGGUUGACCAGAGGGGCAUGC
ACUGUGUUCACCCUGUGGGCCACCUAGUCACCA 148
ACCCU
miR-135a-1 AGGCCUCGCUGUUCUCUAUGGCUUUUUAUUCCU
AUGUGALTUCUACUGCUCACUCAUAUAGGGAUUG 149
GAGCCI3UGGCGCACGGCGGGGACA =
miR-135a-2 AGAUAAAUUCACUCUAGUGCUUUAUGGCUUUUU
(miR-135-2) AUUCCUAUGUGAUAGUAAUAAAGUCUCAUGUAG 150
GGAUGGAAGCCAUGAAAUACAUUGUGAAAAAUC
A
miR-135 CUAUGGC UUUUUAUUCCUAUGUGAUUCLTACUGC
UCACUCAUAUAGGGALTUGGAGCCGUGG 151
miR-135b CACUCUGCUGTJGGCCUAUGGCUUUUCAUUCCUA
UGUGAUUGCUGUCCCAAACUCAUGUAGGGCUAA 152
AAGCCAUGGGCUACAGUGAGGGGCGAGCUCC
miR-136-1 UGAGCCCUCGGAGGACUCCATJUUGUUUUGAUGA
UGGAUUCUUAUGCUCCAUCAUCGUCUCAAAUGA 153
GUCUUCAGAGGGTJUCU
miR-136-2 GAGGACUCCAUUUGUUUUGAUGAUGGAUUCUTJA
UGCUCCAUCAUCGTJCUCAAAUGAGUCUUC 154
miR-137 CUUCGGUGACGGGUALTUCUUGGGUGGAUAAUAC
GGA'QUACGUUGTJTJA1UUGCUUAAGAAUACGCGUA 155
GUCGAGG
miR-138-1 CCCUGGCAUGGUGUGGUGGGGCAGCUGGUGUUG
UGAAUCAGGCCGUUGCCAAUCAGAGAACGGCUA 156
=
CUUCACAACACCAGGGCCACACCACACUACAGG
23
=

CA 02811486 2013-03-26
Precursor Sequence (5' To SEQ ID
Name NO.
miR-138-2 CGUUGCUGCAGCUGGUGUUGUGAAUCAGGCCGA
CGAGCAGCGCAUCCUCUUACCCGGCUAUUUCACG 157
ACACCAGGGUUGCAUCA
miR-138 CAGCUGGUGUUGUGAAUCAGGCCGACGAGCAGC
GCAUCCUCUUACCCGGCUAUUUCACGACACCAGG 158
GUUG
miR-139 GUGUAUUCUACAGUGCACGUGUdUCCAGMTJGG
CUCGGAGGCUGGAGACGCGGCCCUGUUGGAGUA 159
AC
miR-140 UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUA
CCCUAUGGUAGGUIJACGUCAUGCUGUUCUACCA 160
CAGGGUAGAACCACGGACAGGAUACCGGGGCAC
miR-140as UCCUGCCAGUGGUUUUACCCUAUGGUAGGUUAC
GUCAUGCUGUUCUACCACAGGGUAGAACCACGG 161
ACAGGA
miR-140s - CCUGCCAGUGGUUUUACCCUAUGGUAGGUUACG
UCAUGCUGUUCUACCACAGGGUAGAACCACGGA 162
CAGG
rniR-141-1 CGGCCGGCCCUGGGUCCAUCUUCCAGUACAGUG
LTUGGAUGGUCUAAUUGUGAAGCUCCUAACACUG 163
UCUGGUAAAGAUGGCUCCCGGGUGGGUUC
miR-141-2 GGGUCCAUCLTUCCAGUACAGUGLTUGGAUGGUCU
AAUUGUGAAGCUCCUAACACUGUCUGGUAAAGA 164
UGGCCC
ndR-142 ACCCAUAAAGUAGAAAGCACUACUAACAGCACU
GGAGGGUGUAGUGUUUCCUACLTUUAUGGAUG 165
miR-143-1 GCGCAGCGCCCUGUCUCCCAGCCUGAGGUGCAGU
GCUGCAUCUCUGGUCAGUUGGGAGUCUGAGAUG 166
AAGCACUGUAGCUCAGGAAGAGAGAAGUUGUUC
UGCAGC
miR-143-2 CCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUG
GGAGUCUGAGAUGAAGCACUGUAGCUCAGG 167
miR-144-1 UGGGGCCCUGGCUGGGAUAUCAUCAUAUACUGU
AAGUUUGCGAUGAGACACUACAGUAUAGAUGAU 168
GUACUAGUCCGOGCACCCCC
miR-144-2 GGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCG
AUGAGACACUACAGUAUAGAUGAUGUACUAGUC 169
miR-145-1 CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAA
UCCCUUAGAUGCUAAGAUGGGGALTUCCUGGAAA 170
UACUGUUCUUGAGGUCAUGGUU
miR-145-2 CUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGA
UGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCU 171
UGAG
24

CA 02811486 2013-03-26
= =
Precursor Sequence (5 To 3')*
SEQ IlD
Name NO_
miR-146-1 CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUU
CCAUGGGUUGUGUCAGUGUCAGACCUCUGAAAU 172
UCAGUUCUUCAGCUGGGAUAUCUCUGUCAUCGU
miR-146-2 AGCUUUGAGAACUGAALTUCCAUGGGLTUGUGUCA
GUGUCAGACCUGUGAAAUUCAGUUCUUCAGCU 173
rniR-147 AAUCUAAAGACAACAUUUCUGCACACACACCAG
ACUAUGGAAGCCAGUGUGUGGAAAUGCUUCUGC 174
UAGAUU
miR-148a GAGGCAAAGUUCUGAGACACUCCGACUCUGAGU. -
(miR-148) AUGAUAGAAGUCAGUGCACUACAGAACUUUGUC 175
UC
miR-148b CAAGCACGAUUAGCALTUUGAGGUGAAGUUCUGU
UAUACACUCAGGCUGUGGCUCUCUGAAAGUCAG 176
UGCAUCACAGAACUUUGUCUCGAAAGCUUUCUA
miR-148b- AAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUU
small AUACACUCAGGCUGUGGCUCUCUGAAAGUCAGU 177
GCAU
miR-149-1 GCCGGCGCCCGAGCUCUGGCUCCGUGUCUTICACU
CCCGUGCLTUG'UCCGAGGAGGGAGGGAGGGACGG 178
= GGGCUGUGCUGGGGCAGCUGGA
miR-149-2 GCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGUC
CGAGGAGGGAGGGAGGGAC 179
miR-150-1 CUCCCCAUGGCCCUGUCUCCC.AACCCUUGUACCA
GUGCUGGGCUCAGACCCUGGUACAGGCCUGGGG 180
= GACAGGGACCUGGGGAC
miR-150-2 CCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCU
CAGACCCUGGUACAGGCCUGGGGGACAGGG 181
miR-151 UUUCCUGCCCUCGAGGAGCUCACAGUCUAGUAU
GUCUCAUCCCCUACUAGACUGAAGCUCCUUGAG 182
GACAGG
miR-151-2 CCUGUCCUCAAGGAGCUUCAGUCUAGUAGGGGA
UGAGACAUACUAGACUGUGAGCUCCUCGAGGGC 183
AGO
miR-152-1 UGUCCCCCCCGGCCCAGGUUCUGUGAUACACUCC
GACUCGGGCUCUGGAGC.AGUCAGUGCAUGACAG 184
AACUUGGGCCMGAAGG-ACC
miR-1 52-2 GGCCCAGGUUCUGUGAUACACUCCGACUCGGGC
UCUGGAGCAGUCAGUGCAUGACAGAACUUGGGC 185
CCCGG
miR-153-I-1 CUCACAGCUGCCAGUGUCAUUUUUGUGAUCUGC
AGCUAGUAUUCUCACUCCAGUUGCAUAGUCACA 186
AAAGUGAUCAUUGGCAGGUGUGGC
miR-153-1-2 UCUCUCUCUCCCUCACAGCUGCCAGUGUCAUUGU
= CACAAAAGUGAUCAUUGGCAGGUGUGGCUGCUG 187
CAUG
=

CA 02811486 2013-03-26
=
. .
=
Precursor Sequence (5' To 31)* SEQ ID
Name NO.
miR-153-2-1 AGCGGTJGGCCAGUGUCAUUUUUGUGAUGLTUGCA
GCUAGUAAUAUGAGCCCAGUUGCAUAGUCACAA 188 =
AAGUGAUCAUUGGAAACUGUG
miR-1 5 3-2-2 CAGUGUCAUULTUUGUGAUGUUGCAGCUAGUAAli
A UGAGCCCAGUUGCAUAGUCACAAAAGUGAUCA 189
UUG
mil?-1 5 4-1 GUGGUACUUGAAGAUAGGUUAUCCGUGUUGCCU
UCGCUUUAUUUGUGACGAAUCAUACACGGUUGA 190
CCUAUUUUUCAGUACCAA
miR- 154-2 GAAGAUAGGUUAUCCGUGUUGCCUUCGC UUUAU
UUGUGACGA.AUCAUACACGGUUGACCUAUUUUU 191
miR-155 CUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCC
UCCAACUGACUCCUACAUAUUAGCAUUAACAG 192
miR-15 6 = CCUAACACUGUCUGGUAAAGAUGGCUCCCGGGU
miR- GGGUUCUCUCGGCAGUAACCUUCAGGGAGCCCU 193
157=overlap GAAGACCAUGGAGGAC
mil?-14 1
rniR-15 8- GCCGAGACCGAGUGCACAGGGCUCUGACCUAUG
small = miR- AAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGC 194
192 UGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCU
CAAUGCCAGC
mil?- 159-1- UCCCGCCCCCUGUAACAGCAACUCCAUGUGGAAG
small UGCCCACUGGUUCCAGUGGGGCUGCUGUUAUCU 195
GGGGCGAGGGCCA
mil?- 1 61- AAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGG
small UGACUGGUCUGGGCUACGCUAUGCUGCGGCGCU 196
CGGG
miR-1 63-lb- CAUUGGCCUCCUAAGCCAGGGAUUGUGGGLTUCG
small AGUCCCACCCGGGGUAAAGAAAGGCCGAAUU 197
miR-I 63-3- CCUAAGCCAGGGALTUGUGGGUUCGAGUCCCACC
small UGGGGUAGAGGUGAAAGUUCCUUUUACGGAAUU 198
UULTU
miR-162 CAAUGUCAGCAGUGCCUUAGCAGCACGUAAAUA
UUGGCGUUAAGAUUCUAAAAUUAUCUCCAGUAU 199
UAACUGUGCUGCUGAAGUAAGGUUGACCAUACU
CUACAGLTUG
miR-175- GGGCUUUCAAGUCACUAGUGGUUCCG-UUUAGUA
smali=miR- GAUGAUUGUGCAUUGUUUCAAAAUdGUGCCCUA 200
224 GUGACUACAAAGCCC
miR- 177- ACGCAAGUGUCCUAAGGUGAGCUCAGGGAGCAC
small AGAAACCUCCAGUGGAACAGAAGGGCAAAAGCU 201
CAUU
miR-180- CAUGUGUCACUUUCAGGUGGAGUUUCAAGAGUC
small CCUUCCUGGUUCACCGUCUCCUUUGCUCUUCCAC 202
AAC
26

CA 02811486 2013-03-26
Precursor Sequence (5' To 31)* SEQ ID
Name NO.
miR- 181a AGAAGGGCUAUCAGGCCAGCCULTCAGAGGACUC
CAAGGAACAUUCAACGCUGUCGGUGAGUUUGGG 203
AUUUG-AAAAAACCACUGACCGUUGACUGUACCU
UGGGGUCCUUA
miR-18 lb-1 CCUGUGCAGAGAUUATJUUUUUAAAAGGUCACAA
UCAACAUUCAUUGCUGUCGGUGGGUUPAACUGU 204
GUGGACAAGCUCACUGAACAAUGAATJGCAACUG
UGGCCCCGCUU
miR-181b-2 CUGAUGGCUGCACUCAACAUUCAUUGCOGUCGG
UGGGUUUGAGUCUGAAUCAACUCACUGAUCAAU 205
GAAUGCAAACUGCGGACCAAACA
miR-181c CGGAAAAUUUGCCAAGGGUUUGGGGGAACAUUC
AACCUGUCGGUGAGUUUGGGCAGCUCAGGCAAA 206
CCAUCGACCGUUGAGUGGACCCUGAGGCCUGGA
AUUGCCAUCCU
miR-1 82-as GAGCUGCUUdCCUCCCCCCGUUUUUGGCAAUGG
UAGAACUCACACUGGUGAGGUAACAGGAUCCGG 207
UGGUUCUAGACLTUGCCAACUAUGGGGCGAGGAC
UCAGCCGGCAC
miR-182 UUULJUGGCAAUGGUAGAACUCACACUGGU.GAGG
UAACAGGAUCCGGUGGUUCTJAGACUUGCCAACU 208
AUGG
miR-183 CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCA
CUGGUAGAAUUCACUGUGAACAGUCUCAGUCAG- 209
UGAAUUACCGAAGGGCCAUAAACAGAGCAGAGA
CAGAUCCACGA
miR-184-1 CCAGUCACGUCCCCUUAUCACUUUUCCAGCCCAG
CUUUGUGACUGUAAGUGLTUGGACGGAGAACUGA 210
UAAGGGUAGGUGAUUGA
miR-184-2 CCUUAUCACUUUUCCAGCCCAGCUUUGUGACUG
UAAGUGUUGGACGGAGAACUGAUAAGGGUAGG 211
miR- 185-1 AGGGGGCGAGGGAUUGGAGAGAAAGGCAGUUCC
UGAUGGUCCCCUCCCCAGGGGCUGGCUUUCCUCU 212
GGUCCUUCCCUCCCA
miR-185-2 AUGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUC
CCCUCCCCAGGGGCUGGCUUUCCUCUGGUCCUU 213
miR-186-1 ' UGCUUGUAACUUUCCAAAGAALTUCUCCUUUUGG
GCUUUCUGGUTJUUAUUUUAAGCCCAAAGGUGAA 214
UUUUUUGGGAAGULTUGAGCU
miR-186-2 ACUUUCCAAAGAAUUCUCCULTUUGGGCUUUCUG
GUUUUAUUUUAAGCCCAAAGGUGAAUULTUUUGG 215
GAAGU
=
= 27

CA 02811486 2013-03-26
. .
Precursor Sequence (5' To 31)* SEQ ID
Name NO.
miR-187 GGUCGOGCUCACCAUGACACAGUGUGAGACUCG
GGCUACAACACAGGACCCGGGGCGCUGCUCUGA 216
CCCCUCGUGUCUUGUGUUGCAGCCGGAGGGACG
CAGGUCCGCA
miR- 188-1 UGCUCCCUCUCUCACAUCCCUUGCAUGGUGGAG
GGUGAGCUUUCUGAAAACCCCUCCCACAUGCAG 217
GGUUUGCAGGAUGGCGAGCC
miR- 188-2 UCUCACAUCCCUUGCAUGGUGGAGGGUGAGCUU
UCUGAAAACCCCUCCCACAUGCAGGGUUUGCAG 218
GA
miR-J 891 CUGUCGAUUGGACCCGCCCUCCGGUGCCUACUGA
GCUGAUAUCAGUUCUCAULTUUACACACUGOCUC 219
AGUUCAGCAGGAACAGGAGUCGAGCCCUUGAGC
AA
miR- 189-2 CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCA
UULTUACACACUGGCUCAGUUCAGCAGGAACAGG 220
AG
miR- 190-1 UGCAGGCCUCUGUGUGAUAUGUUUGALTAUAUUA
GGUITGUUAUUUAAUCCAACUAUAUAUCAAACAU 221
AUUCCUACAGUGUCUUGCC
miR- 1 90-2 CUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAU
UUAAUCCAACUAUAUAUCAAACAUAUUCCUACA 222
miR-191 -1 CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGC
AGCUGTJUGUCUCCAGAGCAUUCCAGCUGCGCUU 223
GGAUUUCGUCCCCUGCUCUCCUGCCU
miR-191 -2 AGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGU
CUCCAGAGCAUUCCAGCUGCGCUUGGAUUUCGU 224
CCCCUGCU
miR-19 2-2/3 CCGAGACCGAGUGCACAGGGCUCUGACCUAUGA
ALTUGACAGCCAGUGCUCUCGUCUCCCCUCUGGCU 225
GCCAAUUCCAUAGGUCACAGGUAUGLTUCGCCUC
AAUGCCAG
miR-192 GCCGAGACCGAGUGCACAGGGCUCUGACCUAUG
AAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGC 226
UGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCU
CAAUGCCAGC
miR- 1 93-1 = CGAGGAUGGGAGCUGAGGGCUGGGUCLTUUGCGG
GCOAGAUGAGGGUGUCGGAUCAACUGGCCUACA 227
AA.GUCCCAGUUCUCGGCCCCCG
miR-193-2 GCUGGGUCUUUGCGGGCGAGAUGAGGGUGUCGG
AUCAACUGGCCUACAAAGUCCCAGU 228
miR-194-1 AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGU
GGACUGUGUACCAAUUTJCCAGUGGAGAUGCUGU 229
UACULAJUGAUGGUUACCAA
28

CA 02811486 2013-03-26
Precursor Sequence (5' To 31)*
SEQ ID
Name NO.
miR-194-2 GUGUAACAGCAACUCCAUGUGGACUGUGUACCA
AUUUCCAGUGGAGAUGCUGUUACUUUUGAU 230
miR-195-1 AGCUUCCCUGGCUCUAGCAGCACAIGAAATJAUUG
GCACAGGGAAGCGAGUCUGCCAAUATJUGGCUGU 231 =
GCUGCUCCAGGCAGGGUGGUG
=
miR-195-2 UAGCAGCACAGAAAUAUUGGCACAGGGAAGCGA
GUCUGCCAAUAUUGGCUGUGCUGCU 232
miR-196-1 CUAGAGCUUGAAUUGGAACUGCUGAGUGAAUTJA
GGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGA 233
ACACAACAACAUUAAACCACCCGAUUCACGGCA
GUUACUGCUCC
miR-196a-1 GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGG
GUUUCUGAACACAACAACAUUAAACCACCCGAU 234
UCAC
miR-196a-2 UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUU
(miR-196-2) UCAUGUUGUUGGGAUUGAGUUUUGAACUCGGCA 235
ACAAGAAACUGCCUGAGUUACAUCAGUCGGUUU
UCGUCGAGGGC
miR-196 GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGG
GUUUCUGAACACAACAACAUUAAACCACCCGAU 236
= UCAC
miR-196b ACUGGUCGGUGAUUUAGGUAGUUUCCUGUUGUU
GGGAUCCACCTJUUCUCUCGACAGCACGACACUGC 237
CULTCAUUACUUCAGUUG
miR-197 GGCUGUGCCGGGUAGAGAG-. GGCAGUGGGAGGUA
AGAGCUCUUCACCCUUCACCACCUUCUCCACCCA 238
GCAUGGCC
miR-197-2 GUGCAUGUGUAUGUAUGUGUGCAUGUGCAUGUG
UAUGUGUAUGAGUGCAUGCGUGUGUGC 239
miR-198 UCATJUGGUCCAGAGGGGAGAUAGGUUCCUGUGA
UUUUUCCUUCUUCUCUAUAGAAUAAAUGA 240
miR-199a-1 GCCAACCCAGUGUUCAGACUACCUGUUCAGGAG
GCUCUCAAUGUGUACAGUAGUCUGCACAUUGGU 241
UAGGC
miR-199a-2 AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCC
AGUGUUCAGACUACCUGTJUCAGGACAAUGCCGU 242
UGUACAGUAGUCUGCACAUUGGUUAGACUGGGC
AAGGGAGAGCA
rniR-199b CCAGAGGACACCUCCACTJCCGUCUACCCAGUGUU
UAGACUAUCUGUUCAGGACUCCCAAAUUGUACA 243
GUAGUCUGCACAUUGGUUAGGCUGGGCUGGGUU
AGACCCUCGG
miR-199s GCCAACCCAGUGUUCAGACUACCUGUUCAGGAG
GCUCUCAAUGUGUACAGUAGUCUGCACAUUGGU 244
= UAGGC =
29

CA 02811486 2013-03-26
=
=
Precursor Sequence (5' To 3)* SEQ ID
Name NO.
miR-200a GCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGG =
AGUCAGGUCUCUAAUACUGCCUGGUAAUGAUGA 245
CGGC
miR-200b CCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGC
AGCALTUGGAUGGAGUCAGGUCUCUAAUACUGCC 246
UGGUAAUGAUGACGGCGGAGCCCUGCACG
miR-200c CCCUCGUCUUACCCAGCAGUGUUUGGGUGCGGU
UGGGAGUCUCUAAUACUGCCGGGUAAUGAUGGA 247
GO =
miR-202 GLTUCCUUUUUCCUAUGCAUAUACUTJCUUUQAGG
AUCUGGCCUAAAGAGGUAUAGGGCAUGGGAAGA 248
UGGAGC
miR-203 GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUU
CUUAACAGUUCAACAGUUCUGUAGCGCAAUUGU 249
GAAAUGUUUAGGACCACUAGACCCGGCGGGCGC
GGCGACAGCGA
miR-204 GGCUACAGUCUUUCUUCAUGUGACUCGUGGACU
UCCCUUUGUCAUCCUAUGCCUGAGAAUAUAUGA 250
AGGAGGCUGGGAAdGCAAAGGGACGUUCAAUUG
UCAUCACUGGC
miR-205 AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUG
UCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAA 251
CCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAU
GGAGCUGACA
miR-206.-1 UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCC
AUAUGGAUUACUUUGCUAUGGAAUGUAAGGAAG 252
UGUGUGGUUUCGGCAAGUG
miR-206-2 AGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAU
UACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGU 253
UUU
miR-208 UGACGGGCGAGCUUUUGGCCCGGGUUAUACCUG
AUGCUCACGUAUAAGACGAGCAAAAAGCUUGUU 254
GGUCA
miR-210 ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCU
GCCCACCGCACACUGCGCUGCCCCAGACCCACUQ 255
UGCGUGUGACAGCGGCUGAUCUGUGCCUGGGCA.
GCGCGACCC
miR-211 UCACCUGGCCAUGUGACUUGUGGGCUUCCCUUU 256
GUCAUCCUUCGCCUAGGGCUCUGAGCAGGGCAG
GGACAGCAAAGGGGUGCUCAGUUGUCACUUCCC
ACAGCACGGAG
miR-212 COGGGCACCCCGCCCGGACAGCGCGCCGGCACCU
UGGCUCUAGACUGCUUACUGCCCGGGCCGCCCUC 257
AGUAACAGUCUCCAGUCACGGCCACCGACGCCUG
GCCCCGCC
=

CA 02811486 2013-03-26
=
Precursor Sequence (5'.To 31)* SEQ ID
Name NO.
mil?-213-2 CCUGUGCAGAGALTUAUUUUUUAAAAGGUCACAA
UCAA.CAUUCAUUGCUGUCGGUGGGUUGAACUGU 258
GUGGACAAGCUCACUGAACAAUGAAUGCAACUG
UGGCCCCGCUU
miR-213 GAGUUUUGAGGLTUGCUUCAGUGAACAUUCAACG
CUGUCGGUGAGUUUGOAAUUAAAAUCAAAACCA 259
UCGACCGUUGAUUGUACCCUAUGGCUAACCAUC
= AUCUACUCC
miR-214 GGCCUGGCUGGACAGAGUUGUCAUGUGUCUGCC
UGUCUACACUUGCUGUGCAGAACAUCCGCUCAC 260
CUGUACAGCAGGCACAGACAGGCAGUCACAUGA
CAACCCAGCCU
miR-215 AUCAUUCAGAAAUGGUAUACAGGAAAAUGACCU
AUGAAUUGACAGACAAUAUAGCUGAGUUUGUCU 261
GUCAUUUCUUUAGGCCAAUAUUCUGUAUGACUG
UGCUACLTUCAA
miR-216 GAUGGCUGUGAGUUGGCUUAAUCUCAGCUGGCA
ACUGUGAGAUGUUCAUACAAUCCCUCACAGUGG 262
UCUCUGGGAUUAUGCUAAACAGAGCAATJUUCCU =
AGCCCUCACGA
miR-217 AGUAUAAUUAUUACAUAGUUUUUGAUGUCOCAG
AUACUGCAUCAGGAACUGAUUGGAUAAGAAUCA 263
= GUCACCAUCAGUUCCUAAUGCAUUGCCUUCAGC
AUCUAAACAAG
= miR-218-1 = GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUG
AUCUAACCAUGUGGLTUGCGAGGUAUGAGUAAAA 264
CAUGGUUCCGUCAAGCACCAUGGAACGUCACGC
AGCUUUCUACA
miR-218-2 GACCAGUCGCUGCGGGGCUUUCCUUUGUGCTJCTG
AUCUAA CCAUGUGGUGGAACGAUGGAAACGGAA 265
CAUGGUUCUGUCAAGCACCGCGGAAAGCACCGU
GCUCUCCUGCA
miR-219 CCGCCCCGGGCCGCGGCUCCUGALJUGUCCAAACG
CAAUUCUCGAGUCUAUGGCTJCCGGCCGAGAGUU 266
GAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACC
UCGAGCGGG
miR-219-1. CCGCCCCGGGCCGCGGCUCCUGATJUGUCCAAACG
CAAUUCUCGAGUCUAUGGCUCCGGCCGAGAGUU 267
GAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACC
UCGAGCGGG
miR-219-2 ACUCAGGGGCUUCGCCACUGAUUGUCCAAACGC
AAUUCUUGUACGAGUCUGCGGCCAACCGAGAAU 268
UGUGGCUGGACAUCUGUGGCUGAGCUCCGGG
31=

CA 02811486 2013-03-26
=
Precursor Sequence (5' To 31)* SEQ ID
Name
miR-220 GACAGUGUGGcAUUGUAGGGCUCCACACCGUAU
CUGACACUUUGGGCGAGGGCACCAUGCUGAAGG 269
UGUUCAUGAUGCGGUCUGGGAACUCCUCACGGA
_ UCUUACUGAUG
miR-221 UGAACAUCCAGGITCUGGGGCAUGAACCUGGCAU
ACAAUGUAGAUUUCUGUGUUCGUUAGGCAACAG 270
CUACAUUGUCUGCUGGGUUUCAGGCUACCUGGA
AACAUGUUCITC
rniR-222 GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUC
AGUAGCCAGUGUAGAUCCUGUCUUUCGUAAUCA 271
GCAGCUACAUCUGGCUACUGGGUCUCUGAUGGC
AUCUUCUAGCU
miR-223 CCUGGCCUCCUGCAGUGCCACGCUCCGIJGUATJIJTJ
GACAAGCUGAGUUGGACACUCCAUGUGGUAGAG 272
UGUCAGUUUGUCAAAUACCCCAAGUGCGGCACA
UGCUUACCAG
miR-224 GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUA
GAUGAUUGUGCAUUGUUUCAAAAUGGUGCCCUA 273
GUGACUACAAAGCCC
Precursor Sequence (5' To 3')* SEQ ID
Name - NO.
miR-294-1 CAAUCUUCCUUUAUCAUGGUAUUGAUCTUUUCAQ
(chr16) UGCUUCCCUUUUGUGUGAGAGAAGAUA 274
iniR-296 AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGU
UGUGCCUAAUUCAGAGGGLIUGGGUGGAGGCUCU 275
CCUGAAGGGCUCU
miR-299 AAGAAAUGGUUUACCGUCCCACAUACAULTUUGA
AUAUGUAUGUGGGAUGGUAAACCGCUUCUU 276
miR-301 ACUGCUAACGAAUGCUCUGACUUUAUUGCACUA
CUGUACUUUACAGCUAGCAGUGCAAUAGUAUUG 277
UCAAAGCAUCUGAAAGCAGG
miR-302a CCACCACUUAAACGUGGAUGUACUUGCUUUGAA.
ACUAAAGAAGUAAGUGCUUCCAUGUUUUGGU9A 278
UGG
miR-302b GCUCCCUUCAACUUUAACAUGGAAGUGCUtTUCU =
GUGACUUUAAAAGUAAGUGCUUCCAUGUUUTJAG 279
UAGGAGU
miR-302c CCUUUGCUUUAACAUGGGGGUACCUGCUGUGUG
AAACAAAAOUAAGUGCUUCCAUGUUUCAGUGGA 280
GG
miR-302d CCUCUACUUUAACAUGGAGGCACUUGCUGUGAC
AUGACAAAAAUAAGUGCUUCCAUGUUUGAGUGU 281
GG
32

CA 02811486 2013-03-26
Precursor Sequence (5' To 31)*
SEQ ID
=
Name
NO.
miR-320 GCUUCGCUCCCCUCCGCCUTJCUCUUCCCGGUUCU _
UCCCGGAGUCGGGAAAAGCUGGGUUGAGAGGGC 282
GAAAAAGGAUGAGGU
miR-321 UUGGCCUCCUAAGCCAGGGAUUGUGGGUUCGAG
UCC CACC C GGGGUAAAGAAAGGCC GA
283
mi12-323 UUGGUACUUGGAGAGAGGUG-GUCCGUGGCGCGU
= UCGCUUUALTUUAUGGCGCACAUUACACGGUCGA 284
CCUCUUUGCAGUAUCUAAUC
miR-324 CUGACUAUGC CLIC CCC GCAUCCCCUAGGGCAUUG
GUGUAAAGCUGGAGACCCACUGCCCCAGGUGCU 285
GCUGGGGGI.JUGUAGUC
rniR-325 AUACAGUGCUUGGUUCCUAGUAGGUGUCCAGUA
AGUGUUUGUGACAUAAUUUGUUUAUUGAGGACC 286
UCCUAUCAAUCAAGCACUGUGCUAGGCUCUGG
miR-326 CUCAUCUGUCUGUUGGGCUGGAGGCAGGGCCUU
LIGUGAAGGCGGGUGGUGCUCAGAUCGCCUCUGG 287
GCCCUUCCUCCAGCCCCGAGGCGGAUUCA
= rniR-328 UGGAGUGGGGGGGCAGGAGGGOCUCAGGGAGAA
AGUGCAUACAGCCCCUGGCCCUCUCUGCCCUUCC 288
GUCCCCUG
miR-330 CUUUGGCGAUCACUGCCUCUCUGGGCCUGUGUC
UUAGGCUCUGCAAGAUCAACCGAGCAAAGCACA 289
CGGCCUGC-AGAGAGGCAGCGCUCUGCCC
miR-331 GAGUUUGGUULTUGUUUGGGITUUGUUCUAGGUAU
GGUCCCAGGGAUCCCAGAUCAAACCAGGCCCCUG 290
GGCCUAUCCUAGAACCAACCUAAGCUC
miR-335 UGUUUUGAGCGGGGGUCAAGAGCAAUAACGAAA.
AAUGUUUGUCAUAAACCGUUUUUCAUUAbUGCU 291
CCUGACCUCCUCUCAUUUGCUAUAUUCA
miR-3 3 7 GUAGUCAGUAGTJUGGGGGGUGGGAACGGCUUCA
UACAGGAGLTUGAUGCACAGUUAUCCAGCUCCUA 292
UAUGAUGCCUUUCUUCATJCCCCUUCAA
miR-338 UCUCCAACAAUAUCCUGGUGCUGAGUGAUGACU
CA G GC GACUC CAG CAUCAGUGAUUUUGUUGAAG 293
A
miR-339 CGGGGCGGCCGCUCUCCCUGUCCUCCAGGAGCTIC
ACGUGUGCCUGCCTJGUGAGCGCCTJCGACGACAG 294
= AGCCGGCGCCUGCCCCA GUGUCUGCGC
n2iR-340 UUGUACCUGGUGUGAUUAUAAAGCAAUGAGACU
GAUUGUCAUAUGUCGUUUGUGGGAUCCGUCUCA= 295
GUUACULTUAUAGCCAUACCUGGUAUCUUA
miR-342 GAAACUGGGCUCAAGGUGAGGGGUGCUAUCUGU
GAUUGAGGGACATJGGUUAAUGGAAUUGUCUCAC 296
ACAGAAAUCGCACCCGUCACCUUGGCCUACUUA
=
33

CA 02811486 2013-03-26
Precursor Sequence (5' To 31)* SEQ LD
Name NO.
miR-345 ACCCAAACCCUAGGUCUGCUGACUCCUAGUCCAG
GGCUCGUGAUGGCTJGGUGGGCCCUGAACGAGGG 297
=
GUCUGGAGGCCUGGGUUUGAAUAUCGACAGC
miR-346 GUCUGUCUGCCCGCAUGCCUGCCUCUCUGUUGCU
CUGAAGGA.GGCAGGGGCUGGGCCUGCAGCUGCC 298
UGGGCAGAGCGGCUCCUGC
miR-367 CCAUUACUGUUGCUAAUAUGCAACUCUGUUGAA
UAUAAAUUGGAATJUGCACUUUAGCAAUGGUGAU 299
GG =
miR-368 AAAAGGUGGAUATJUCCUUCUAUGUUUAUGUUAU
UUAUGGUUAAACAUAGAGGAAAUUCCACGULTIEJ 300
miR-369 UUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUU
AUUGACUUCGAAUAAUACAUGGUUGAUCUUUUC 301
UCAG
miR-370 AGACAGAGAAGCCAGGUCACGUCUCUGCAGUUA
CACAGCUCACGAGUGCCUGCUGGGGUGGAACCU 302
GGUCUGUCU
miR-371 GUGGCACUCAAACUGUGGGGGCACUUUCOGCUC
' UCUGGUGAAAGUGCCGCCAUCUUUUGAGUGUUA 303
miR-372 GUGG GC CLICAAAUGUGGAGCACUAUUCUGAUGU
C CAAGUGGAAAGUGCUGCGACAUUUGAGCGUCA 304
miR-373 GGGAUACUCAAAAUGGGGGCGCUUUCCUUUUUG
UCUGUACUGGGAAGUGCUUCGAUUUUGGG GUGU 305
= CCC =
miR-374 UACAUCGGC CAITUAUAAUACAACCUGAUAAGUG
UUAUAGCACUUAUCAGAUUGUAUUGUAAUUGUC 306
UGUGUA
miR-hes1 AUGGAGCUGCUCACCCUGUGGGCCUCAAAUGUG
GAGGAACUAUUCUGAUGUCCAAGUGGAAAGUGC 307
UGCGACAUUUGAGCGUCACCGGUGACGCCCAUA
UCA
miR-hes2 GCAUCC CCUCAGCCUGUGGCACUCAAACUGUGG
GCACUUUCUG CIJ CUCUG GUGAAAGUG C C GC C 308
AUCUUUUGAGUG UUACCGCUUGAGAAGACUCAA
CC
rniR-hes3 CGAGGA:GCUCATJACUGGGAUACUCAAAAUGGGG
GCGCUUUCCUUUUUGUCUGUUACUGGGAAGUGC 309
UUCGAUUUUGGGGUGUCCCUGUUUGAGUAGGGC
AUC
* An underlined sequence within a precursor sequence corresponds to a mature
processed miR transcript (see Table lb). Some precursor sequences have two
34=

SE
di
zr-witu SZE ruStinaga2B-
eart2uoSnm
dg
LI-21fw LZ
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:1-9 flilta 9ZC
SoSnnuneuvrt.SovoacaSun 9 I-N-ltu
SZE uounn.n22-t-
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:DS f-mul 17ZE
2ann.n2STreunuaeoSeaSun vg rlutti
q0/-Yful EZC
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Pt.-Wm
!E-6-2Bui I ZE ruSgev2o3BunpReno5s-
aen 4, 6-?! 2W
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:1-6-11w OZC -
eSnsen2noRenonen.n22nnnon 6-2llui
fur-z-witu 61 nn rc
EnnneSnaen n. o-eSnSa L-zutu
_
z-qt-mui
- f-qr-wita :q[-?ilui 81E vrenen2u82-evvanve2Bn
!L-191 LI noSannnSenan22-an !L-1a1
Ll 9I namenSnnnSenStn22-e2rt z-pi
S IC
narnunSurregengenSau2n iL
az-Jai -17I n.WenvannS2132Ssr[22-132n
=
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en.SenOReSe PL-191
az-m Z I nuntrerenaS-
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qZ7lal TIC
nn2SnStt2n.n2Reti2nneSn U-P1
fr-vZ-191
:Z-vZ-P1 0 I nnOvneannneraeri.22-
e2n
aumm
aiqui aas (s)vt\rdozolu.1 (1 01- .S) VNIiuI
.rosxnoard Sumuodsa.uop 01 bas avaanbos yweip.0 azriluyq axareyq
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aI( marj pan.pap OJimp slum annum iugaaj.sip com. SuIlouop saouonbas pounzapun
9Z-0-ETOZ 98VIT8Z0 VD

95
=
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oas3rtruartuS3on.ae0nrin0 -v0E-vut
v0E-Vta Lt7 E
auunnasSonooneamean -v0E-vut
06Z-Vta 9t E
unaBones=annnuoveo gen a6E-ylui
z-q6Z-Vui -7-46Z-Via ç5
n22Drtusu2nnnsoa-sogen q6Z-Via
v6Z-21.1w !E--v6E-VW trfrE nn22ormanorreoovoguno=
Eff 2unnut-Longsaeono3u22-
eu RE-nur
Z-4LZ-Val ZVE
2nonn2utrrtoBaWeae3nn qz id/
vL E-2/7ufWEoonon-tiV=esno2.2thasaeonn vLe-=-y-p.0
49Z-?17ut n22vn-
e22uDrinvengsvann q9-flu!
Z-719E-Via
1-v9Z-Via !v9E-Vul 6 noasne22-
eoontsnave3nn v9E-ylui
8 EE uUnon223non2nnaeo2nnvo
cz-vut
617Z-VW
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75-E-vut
(1EZ-Wal 95 . asannnu222-
goo2nnu3sons qcz-ypa
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e222oo2nrceavont
. .
Ze-Ylut
n2nozats2nnigvoo2n92nu ZZWJtU
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(DOE-Via) OZ-Ylta "(=; E unRgeo2nRenerina2nReeen
0E-vur
Z-46T-Ylu1 :/-46T-Ww TEE
u2nousgeoareporreveo2n2n q6 plaza
Et 0
g2rouguranenon1Je=e32an no-r-vur
ET-g 8t11W 6ZE
gnarn2rtRunorreo2riBBeUlt g [-via
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¨ =
=
= =
9Z-0-ETOZ 98VIT8Z0 VD

CA 02811486 2013-03-26
Mature Mature raiRNA Sequence SE0 ID Corresponding precursor =
miRNA = (5' to 3') NO. microRNA(s); see Table la
Name
miR-30d uguaaacauccccgacuggaag 351 miR-30d
miR:-30e uguaaacauccuugacugga 352 miR-30e
miR-31 ggcaagaugcuggcanagcug 353 miR-31
miR-32 uauugcacauuacuaaguugc 354 miR-32
miR-33 gugcauuguaguugcauug 355 miR-33; miR-33b
miR-34a uggcagugucuuagcugguugu 356 miR-34a
miR-34b aggcagugucauuagcugauug - 357 miR-34b
miR-34c aggcaguguaguuagcugauug 358 miR-34c
miR-92 uauugcacuugucccggccugu 359 miR-92-2; miR-92-1
miR-93 aaagugcuguucgugcagguag 360 miR-93-1; rniR-93-2
miR-95 uucaacggguauuuauugagca 361 miR-95
miR-96 uuuggcacuagcacauuuuugc - 362 miR-96
ugagguaguaaguuguauuguu - 363 . miR-98 =
miR-99a aacccguagauccgaucuugug - 364 miR-99a
miR-99b cacccguagaaccgaccuugcg - 365 miR-99b
miR-100 uacaguacugugauaacugaag 366 - miR-100
miR-101 uacaguacugugauaacugaag 367 rniR-.101-1; miR-101-2
miR-103 agcagcauuguacagggcuauga .368 miR-J 03-i
miR-105 ucaaauicucagacuccugu 369 miR-105
miR-106- aaaagugcuuacagugcagguagc - 370 mi_R-106-a
a
miR-106- uaaagugcugacagugcagau 371 miR-106-b
miR-107 agcagcauuguacagggcuauca 372 miR-107
37

CA 02811486 2013-03-26
Mature Mature miRNA Sequence SEQ 11) Corresponding precursor
miRNA (5' to 3') NO. microRNA(s); see Table la
Name
miR-122a uggagugugaeaaugguguuugu 373 miR-122a-1; miR-122a-2
miR-124a uuaaggcacgcggugaaugoca 374 m1R-124a-1; miR-124a-2;
miR-124a-3
miR-125a ucccugagacccunuaaccugug 375 miR-125a-1; miR-125a-2
miR-125b ucccugagacccuaacuuguga 376 miR-125b-1; miR-125b-2
miR-126* cauuauuacuuuugguacgcg 377 miR-126-1; miR-126-2
miR-126 ucguaccgugaguaauaaugc 378 rn1R-126-1; miR-126-2
miR-127 ucggauccgucugagcuuggcu 379 miR-127-1; miR-127-2
miR-128a ucacaguga.accggucucuuuu 380 miR-128; miR-128a
mi.R-128b ucacagugaaccggucucuuuc = 381 rniR-1281
miR-129 cuuuuuguggucligggcuagc 382 miR-129-1; miR-129-2
miR-130a cagugcaaugutiaaaagggc = .383 miR-130a
miR-130b cagugcaaugaugaaagggcau 384 miR-130b
miR-132 uaacagucuacagccauggucg 385 miR-132-1
rniR-133a uugguccccuucaaccagcugu 386 miR-133a-1; miR-133a-2
miR-133b uugguccccuucaaccagcua 387 miR-133b
miR- 13 4 ugugacugguugaccagaggg 388 miR-134-1; miR-134-2
miR-135a uauggcuuuuuaunccuauguga 389 miR-135a; miR-135a-2
(miR-135-2)
miR-135b uauggcuuuucauuccuaugug 390 miR-135b
miR-136 acuccauuuguuuugaugaugga 391 miR-136-1; miR-136-2
miR-137 uauugcuuaagaaua.cgeguag 392 miR-137
miR-138 agcuggugmigugaauc 393 miR-138-1; miR-138-2
38

=
6
=
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reart22on2no2anonueort v[gr-gpu
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env2n2oneuno2nuenn ssi-N7ut
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1-t=ST-Ylut I1-17 2onnonanOoorrenn22vn r -
ylut =
Z-Z-EST-Put
==`Z
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Z-ZS :I-E.57-2rN" 6017 annotatopgrreo2ngeon Ecr-
giut
8017 B2gOnnolnEg-anovSnnov
r-zilui
z-os. .7-057-Ylut LOt= = ' n5vao-
eann000-e=noonon r-rut
9017
o3nouo1lnoano3no2Shon 6171-ypa
qgfrI-ZIltu gOt
annnotTamearrea5naeon rigkr-wut
(81711111tu) PRPT-Mul 17017
annrizeuBaenagoOn.tuon vgiu-wut
Z 1-P 017 zfrr-vut
E-9fr1=-.211u1 :1-917I-?But zgi7 nn222nuD3nneanozeg22n
9frr-yitu
E-S171=-=Nlitt :1-SPI-211ut 1017 nn000nuaamonnnnaeoong r-mw
Z.-PPP-Out !E-P17/-Pw 0017 2-enourerteSingettertgeoun
frfr
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dc .
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[p[-via
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017
E-v66 [-Val 9E-17 onnSttoo-
ertouReonan2Euoo t2661-27-mt
861-211u1 SRent-e2E22gReoon22 g6
161-211w 17E-17
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496 T-Y1u1 EE-17
22nn2nnStioonntaert2vti q96 [-put
(E-96.1-117u4
Z.-1'961-211w :696 I-217u1 Z
22nn2nanuortnnEen22Bn 1,961-211w
g6I-Ulta [-Wm I Et- onnnurraesPeoRoBR32m
6 f -Out
E-176 r-fra 017
u22n2nspormaeoggoupten fr6r-?Hw
Z.- 6 I- E61-211tu 6Z-17
.c000nguee3enoo32nove E6 r-Mut
06 I-217ul 8Z17 oogeoannuannroaegn3 E 6T-?I!' _
E-1.6 I-Mtu LZ17 nogeomegooan-ae220-euo
r 6 f-Ylill
Z-061-VW 97 ngann-
enenannanenRgn 061-21-lui
E-687-al7w .7-6RT-W1tu SZ-17 nonunv2-noge2no-onoo2n ar-zaut
88 I-211tu 17Z-17 ng22-022a2n-eann000nuo
r-vut
.L8 I-Vul Ea' 2oogeo2nren2nn3n2n23n
E-98I-211u1! 1.--991-217w ZZ-17
nuo2EStirranoonorareg2eueo 9g T-pw
E-S81:-.21.1w TZ'17 ontauo2Re-m22-
e23n cgr-vut
E-P8I-Y.1111: On7 n22Z-
evnufnovau2o03n frg r-var
ER I-NPLI 6117
SnagonnutaertSameaRnun Eg r-y! it/
svn'T-Zilut !ES' r-N7F-1 8117 -
enomooSnnorgertonnn * r-zput
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qrgi-Aqui
oureN
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am3211
9Z-0-ETOZ 9817TT8Z0 VD

CA 02811486 2013-03-26
=
"= __________________________________________________________________
Mature Mature miRNA Sequence SEQ ID Corresponding precursor
miRNA (5' to 3') NO. microRNA(s); see Table la
Name
miR- uacaguagucugcacauugguu 437 miR-199a-1;
miR-199a-2;
199a* rniR-199s;
mil?-199b
miR-199b cccagilgunuagacuaucuguuc 438 miR-199b
. .
miR-200a uaacacugucugguaacgaugu 439 miR-200a -
nziR-200b cucuaauacugccuggnaaugaug 440 miR-200b
miR-200c aauacugccggguaaugaugga 441 miR-200c
. nziR-202 agagguauagggcauggga.aga 442 miR-202
miR-203 gugaaauguuuaggaccacuag 443 miR-203
mi.R-204 uucccuuugucauccuaugccu 444 miR-204
miR-205 uccuucauuccaccggagucug 445 miR-205
miR-206 uggaauguaaggaagugugugg 446 miR-206-1; miR-206-2
rniR-208 auaagacgagcaaaaagcuugu 447 miR-208
miR-210 cugugcgugugacageggcug 448 miR-210
miR-211 uucccuungucauccuucgccu 449 miR-211
miR-212 uaacagucuccagucacggcc 450 miR-212
miR-2.13 accaucgaccguugauuguacc 451 miR-213
miR-214 acagcaggcacagacaggcag 452 miR-214
miR-215 augaccuaugaauugacagac 453 miR-215
miR-216 uaaucucagcuggcaacugug 454 miR-216
miR-217 uacugcaucaggaacugauuggau 455 miR-217
miR-218 nugugcuugaucuaaccaugu 456 miR-218-1; miR-218-2
miR-219 ugauugnccaaacgcaauucu 457 - miR-219;
miR-219-1;
miR-219-2 =
miR-220 ccacaccguaucugacacuuu 458 miR-220
41

CA 02811486 2013-03-26
-
Mature Mature miRNA Sequence SEQ ID Corresponding precursor
miRNA (5' to 3') NO. microRNA(s); see
Table la
Name
miR-221 agcuacauugucugcuggguuuc 459 miR-221
miR-222 agcuacaucuggcuacUgggucuc . 460 .miR-222
miR-2 2 3 ugucaguuugucaaauacccc 461 miR-223
miR-224 caagucacuagugguuccguuua 462 miR-224
_ .
. miR-2 96 agggcccccccucaauccugu 463 miR-296
miR-299 ugguuuaccgucccacauacau 464 rn1R-299
_ __________________________________________________________________
miR-3Q1 cagugcaauaguauugucaaagc . 465 miR-301
miR-302a uaagugcuuccauguuuugguga 466 miR-302a
_ __________________________________________________________________
miR- acuunaacauggaagugcuuucu = 467 miR-302b
302b* _
miR-302b uaagugcuuccauguuuuaguag 468 miR-302b
miR- uuuaacauggggguaccugcug 469 miR-302c
302c* _ ______________________________
miR-302c uaagugcuuccauguuucagugg 470 miR-302c
miR-302d uaagugcuuccauguuugagugu 471 miR-302d
ImiR-320 aaaagcuggguugagagggcgaa 472 miR-320
miR-321 uaagccagggauuguggguuc 473 miR-321
=
miR-323 gcacau.uacacggucgaccucu 474 miR-323
in iR-324- cgcauccccuagggcauuggugu 475 miR-324
5p
miR-324- ccacugccccaggugcugcugg 476 miR-324
3p
miR-325 ccuaguagguguccaguaagu 477 miR-325
miR-326 ccuCuiggeccuuccuccag 478 miR-326
___________________________________________________________________ _
miR-328 cuggcccucucugcccuuccgu ' 479 miR-328
42

CA 02811486 2013-03-26
= =
Mature Mature rniRNA Sequence SEQ ID Corresponding precursor
miRNA (5' to 3') NO.
microRNA(s); see Table la
Name
iniR-330 geaaagcacaeggccugcagaga 480 miR-330
miR-331 gccceugggccuauectiagaa - 481 miR-331
miR-335 ucaagagcaalia cgaaaaaugu - 482 miR-335
miR-337 uccagcuccuauaugaugccuuu - 483 rniR-337
rniR-338 uccagcaucagugauuuuguuga 484 miR-338
miR-339 ueccuguccuccaggagcuca 485 miR-339
miR-340 uccgucucaguuacuuuauagce - 486 miR-340
miR-342 ucucaeacagaaaucgcacccguc 487 miR-342
miR-345 ugcugacuccuaguccagggc 488 miR-345
L_
miR-346 ugucugcccgcaugccugccucu 489 miR-346
miR-367 aauugcacuuuagcaaugguga 490 miR-367
miR-368 r acauagaggaaauuccacguuu - 491 miR-368
miR-369 aauaauacaugguugaucuuu 492 miR-369
miR-370 gccugcugggguggaaccugg 493 miR-370
miR-371 gugccgccaucuuuugagugu 494 miR-371
rniR-372 aa.agugeugegacauuugagegu 495 miR-372
miR-373* acucaaaaugggggcgcuuuce 496 miR-373
miR-373 gaagugcuucgauuuuggggugu 497 miR-373
miR-374 uuauaauacaaccugauaagug 498 miR-374
=
The present invention encompasses methods of diagnosing whether a subject -
has, or is at risk for developing, a solid cancer, comprising measuring the
level of at
43

CA 02811486 2013-03-26
least one miR gene product in a test sample, from the subject and comparing
the level of
the miR gene product in the test sample to the level of a corresponding miR
gene
product in a control sample. As used herein, a "subject" can be any mammal
that has,
or is suspected of having, a solid cancer. In a preferred embodiment, the
subject is a
human who has, or is suspected of having, a solid cancer.
In one embodiment, the at least one miR gene product measured in the test
sample is selected from the group consisting of miR-21, miR-17-5p, miR-191,
miR-
29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155,
miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c, miR-25,
miR-
221, miR-106a and combinations thereof. In a particular embodiment, the miR
gene
product is miR-21, miR-191 or miR-17-5p. In another embodiment, the miR gene
product is not miR-15a or miR-16-1. In an additional embodiment, the miR gene
product is not miR 159-1 or miR-192. In an additional embodiment, the miR gene
product is not miR-186, miR-101-1, miR-194, miR-215, miR-.106b, miR-25, miR-
93,
miR-29b, miR-29a, miR-96, miR-182s, miR-182as, miR-183, miR-129-1, let-7a-1,
let-
7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-
1,
let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s,
miR-142as, miR-105-1, or miR-175. In a farther embodiment, the miR gene
product is
not miR-21, miR-301, miR-142as, miR-142s, miR-194, miR-215, or miR-32. In
another embodiment, the miR gene product is not miR-148, miR-10a, miR-196-1,
miR-
152, miR-196-2, miR-148b, miR-10b, miR-129-.1, miR-153-2, ma-202, miR-139, let-
7a, let-7f, or let-7d. In yet another embodiment, the miR gene product is not
miR-15a,
miR-16-1, miR-182, miR-181, miR-30, miR-15a, miR-16-1, miR-15b, miR-16-2, miR-
195, miR-34, miR-153, miR-21, miR-217, miR-205, miR-204, miR-143,
miR-96, miR-103, miR-107, miR-129, miR-9, miR-137, miR-217, miR-186.
The solid cancer can be any cancer that arises from organs and solid tissues.
Such cancers are typically associated with the formation and/or presence of
tumor
masses and can be carcinomas, sarcomas and lymphomas. Specific examples of
solid
cancers to be diagnosed by the methods of the invention include, but are not
limited to,
colon cancer, rectal cancer, stomach (gastric) cancer, pancreatic cancer,
breast cancer,
lung cancer, prostate cancer, bronchial cancer, testicular cancer, ovarian
cancer, uterine
cancer, penile cancer, melanoma and other skin cancers, liver cancer,
esophogeal
44

CA 02811486 2013-03-26
cancer, canters of the oral cavity and pharynx (e.g., tongue cancer, mouth
cancer),
cancers of the digestive system (e.g., intestinal cancer, gall bladder
cancer), bone and
joint cancers, cancers of the endocrine system (e.g., thyroid cancer), brain
cancer, eye
cancer, cancers of the urinary system (e.g., kidney cancer, urinary bladder
cancer),
Hodgkin disease and non-Hodgkin lymphoma. In particular embodiments, the solid
cancer is not one or more of breast cancer, lung cancer, prostate cancer,
pancreatic
cancer or gastrointestinal cancer.
In one embodiment, the solid cancer is breast cancer or lung cancer and the at
least one miR gene product measured in the test sample is selected from the
group
consisting of miR-210, miR-213 and a combination thereof.
In a further embodiment, the solid cancer is colon cancer, stomach cancer,
=
prostate cancer or pancreas cancer and the at least one miR gene product
measured in
the test sample is miR-218-2.
In a certain embodiment of the invention, the solid cancer is breast cancer
and
the at least one miR gene product measured in the test sample is selected from
the
group consisting of miR-125b-1, miR-125b-2, miR-145, miR-21 and combinations
thereof. In a related embodiment, the solid cancer is breast cancer and the at
least one
miR gene product in the test sample is selected from the group consisting of
miR-21,
miR-29b-2, miR-146, miR-125b-2, miR-125b-1, miR-10b, miR-145, miR-181a, miR-
140, miR-213, miR-29a prec, miR-181b-1, miR-199b, miR-29b-1, miR-130a, miR-
155,
let-7a-2, miR-205, miR-29c, miR-224, miR-100, miR-31, miR-30c, miR-17-Sp, miR-
210, miR-122a, miR-16-2 and combinations thereof. In a related embodiment, the
solid
cancer is breast cancer and the at least one miR gene product is not miR-15a
or miR-
16-1. In a further embodiment, the solid cancer is breast cancer and the at
least one
miR gene product is not miR-145, miR-21, miR-155, miR-10b, miR-125b-1, rniR-
125b-2, let7a-2, let7a-3, let-7d, miR-122a, miR-191, miR-206, miR-210, let-7i,
miR-
009-1 (miR131-1), miR-34 (miR-170), miR-102 (miR-29b), miR-123 (miR-126), miR-
140-as, miR-125a, miR-194, miR-204, miR-213, let-7f-2, miR-101, rniR-128b, miR-
136, miR-143, miR-149, miR-191, miR-196-1, miR496-2, MiR-202, miR-103-1, or
miR-30c_ In another embodiment, the solid cancer is breast cancer and the miR
gene
product is not miR-21, miR-125b-1, let-la-2, let-7i, miR-100, let-7g, miR-31,
miR-32a-
1, miR-33b, miR-34a-2, miR-101-1, miR-135-1, miR-142as, miR-142s, miR-144, miR-

CA 02811486 2013-03-26
301, miR-29e, miR-30c, miR-106a, or miR-29b-1. In yet another embodiment, the
solid cancer is breast cancer and the miR gene product is not miR-159-1 or miR-
192.
In an additional embodiment, the solid cancer is breast cancer and the miR
gene
=
product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93,
miR-29b, miR-29a, miR-96, miR-182s, miR-182as, miR-183, miR-129-1, let-7a-1,
let-
7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-
1,
let-7a-2, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s,
miR-142as, miR-105-1, or miR-175. In a further embodiment, the solid cancer is
breast cancer and the miR gene product is not miR-21, miR-301, miR-142as, miR-
142s,
miR-194, miR-215, or miR-32. In another embodiment, the solid cancer is breast
cancer and the miR gene product is not miR-148, miR-10a, miR-196-1, miR-152,
miR-
196-2, miR-148b, rniR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a, let-
7f,
or let-7d. In yet another embodiment, the solid cancer is breast cancer and
the miR
gene product is not miR-18th, miR-181c, miR-181d, miR-30, miR-15b, miR-16-2,
miR-153-1, rniR-217, miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or
miR-137.
In another embodiment, the solid cancer is colon cancer and the at least one
rniR gene product in the test sample is selected from the group consisting of
miR-24-1,
miR-29b-2, miR-20a, miR-10a, miR-32, miR-203, miR-106a, miR-1'7-5p, miR-30c,
miR-223, miR-126*, miR-128b, miR-21, miR-24-2, miR-99b prec, miR-155, miR-213,
miR-150, miR-107, miR-191, miR-221, miR-9-3 and combinations thereof. In
another
embodiment, the solid cancer is colon cancer and the miR gene product is not
miR 159-
1 or !I-AR-192. In an additional embodiment, the solid cancer is colon cancer
and the
miR gene product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-
25,
miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182as, miR-183, miR-129-1, let-
7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-
192,
miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301,
miR-142s, miR-142as, miR-105-1, or miR-175. In a further embodiment, the solid
cancer is colon cancer and the miR gene product is not miR-21, miR-301, miR-
142as,
miR-142s, miR-194, rniR-215, or miR-32. In another embodiment, the solid
cancer is
= colon cancer and the miR gene product is not miR-148, miR-10a, miR496-1,
miR-152,
miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139, let-7a,
= 46

CA 02811486 2013-03-26
let-7f, or let-7d. In yet another embodiment, the solid cancer is colon cancer
and the
miR gene product is not miR-181b, miR-181c, miR-181d, miR-30, miR-15b, miR-16-
2,
miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or
miR-137.
In yet another embodiment, the solid cancer is lung cancer and the miR gene
product in the test sample is selected from the group consisting of miR-21,
miR-205,
miR-200b, miR-9-1, miR-210, miR-148, miR-141, miR-132, miR-215, miR-128b, let-
7g, miR-16-2, miR-129-1/2 prec, raiR-126*, miR-142-as, miR-30d, miR-30a-5p,
miR-
7-2, miR-199a-1, miR-127, miR-34a prec, miR-34a, miR-136, miR-202, miR-196-2,
miR-199a-2, let-7a-2, miR-124a-1, miR-149, miR-17-5p, miR-196-1 prec, miR-10a,
miR-99b prec, miR-196-1, miR-199b, miR-191, miR-195, miR-1 55 and combinations
thereof. In a related embodiment, the solid cancer is lung cancer and the at
least one
miR gene product is not miR-15a or miR-16-1. In a further embodiment, the
solid =
cancer is lung cancer and the at least one miR gene product is not miR-21, miR-
191,
miR-126*, miR-210, miR-155, miR-143, miR-205, miR-126, miR-30a-5p, miR-140,
miR-214, miR-218-2, miR-145, miR-106a, miR-192, miR-203, miR-150, miR-220,
miR-192, miR-224, miR-24-2, miR-212, miR-9, miR-17, miR-124a-1, miR-95, miR-
198, miR-216, miR-219-1, miR-197, miR-125a, miR-26a-1, miR-146, miR-199b,
let7a-
2, miR-27b, miR-32, miR-29b-2, miR-33, miR-181c, miR-101-1, miR-124a-3, miR-
1-25b-1 or let7f-1. In another embodiment, the solid cancer is lung cancer and
the at
least one miR gene product is not miR-21, miR-182, miR-181, miR-30, miR-15a,
miR-
143, miR-205, miR-96, miR-103, miR-107, miR.-129, miR-137, miR-186, miR-15b,
miR-16-2, miR-195, miR-34, miR-153, miR-217, miR-204, miR-211, miR-9, miR-217,
let-7a-2 or miR-32. In a further embodiment, the solid cancer is lung cancer
and the
miR gene product is not let-7e, let-7g, miR-7-3, miR-210, miR-31, miR-34a-1,
miR-a- .
2, miR-99a, miR-100, miR-125b-2, miR-132, miR-13571., miR-195, miR-34, miR-
123,
miR-203. In another embodiment, the solid cancer is lung cancer and the miR
gene
product is not miR 159-1 or miR-192. In an additional embodiment, the solid
cancer is
lung cancer and the miR gene product is not miR-186, miR-101-1, miR-194, miR-
215,
.30 miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-
182as, miR-
183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-
32, miR-
159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190,
47

CA 02811486 2013-03-26
miR-21, miR-301, miR-142s, miR-142as, miR-105-1, or miR-175. In a further
embodiment, the solid cancer is lung cancer and the miR gene product is not
miR-21,
miR-301, miR-142as, miR-142s, miR-194, miR-215, or miR-32. In another
embodiment, the solid cancer is lung cancer and the miR gene product is not
miR-148,
miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-
153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another embodiment,
the
solid cancer is lung cancer and the miR gene product is not miR-181b, miR-
181c, miR-
181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-
103, miR-107, miR-129-2, miR-9 or miR-137. =
= 10 In a further embodiment, the solid cancer is pancreatic cancer
and the at least
one miR gene product measured in the test sample is selected from the group
consisting
of miR-103-1, miR-103-2, miR-1 55, miR-204 and combinations thereof. In a
related
embodiment, the solid cancer is pancreatic cancer and the miR gene product in
the test
sample is selected from the group consisting of miR-103-2, miR-103-1, miR-24-
2,
miR-107, miR-100, miR-125b-2, miR-125b-1, miR-24-1, miR-191, miR-23a, miR-26a-
1, miR-125a, miR-130a, miR-26b, miR-145, miR-221, miR-126'', miR-16-2, miR-
146,
miR-214, miR-99b, miR-128b, miR-155, miR-29b-2, miR-29a, miR-25, miR-16-1,
miR-99a, miR-224, miR-30d, miR-92-2, miR-199a-1, miR-223, miR-29c, miR-30b,
miR-129-1/2, miR-197, miR-17-5p, miR-30c, miR-7-1, miR-93-1, miR-140, miR-30a-
5p, miR-132, miR-181b-1, miR-152 prec, miR-23b, miR-20a, miR-222, miR-27a, miR-
92-1, miR-21, miR-129-1/2 prec, miR-150, miR-32, miR-106a, miR-29b-1 and
combinations thereof. In one embodiment, the solid cancer is pancreatic cancer
and the
miR gene product is not miR-15a or miR-16-1. In another embodiment, the solid
cancer is pancreatic cancer and the miR gene product is not miR 159-1 or miR-
192. In
an additional embodiment, the solid cancer is pancreatic cancer and the miR
gene
product is not miR-186, miR-101-1, miR-194, miR-215, miR-106b, miR-25, miR-93,
miR-29b, miR-29a, miR-96, miR-182s, miR-182as, miR-183, miR-129-1, let-7a-1,
let-
7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-32, miR-159-1, miR-192, miR-125b-
1,
let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-21, miR-301, miR-142s,
miR-142as, miR-105-1, or miR-175. In a further embodiment, the solid cancer is
pancreatic cancer and the miR gene product is not miR-21, miR-301, miR-142as,
miR-
142s, miR-194, miR-215, or miR-32. In another embodiment, the solid cancer is
48

CA 02811486 2013-03-26
pancreatic cancer and the miR gene product is not miR-148, miR-10a, miR-196-1,
miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-139,
let-7a, let-7f, or let-7d. In yet another embodiment, the solid cancer is
pancreatic
cancer and the miR gene product is not miR-181b, miR-181c, miR-181d, miR-30,
miR-
15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107, miR.-
129-2, miR-9 or raiR-137.
In another embodiment, the solid cancer is prostate cancer and the miR gene
product in the test sample is selected from the group consisting of let-7d,
miR-128a
prec, miR-195, miR-203, let-7a-2 prec, miR-34a, miR-20a, miR-218-2, miR-29a,
miR-
25, miR-95, miR-197, miR-135-2, miR-187, miR-196-1, miR-148,
let-7i, miR-198, miR-199a-2, miR-30c, miR-17-5p, miR-92-2, miR-146, miR-181b-1
prec, miR-32, miR-206, miR-184 prec, miR-29a prec, miR-29b-2, miR-149, miR-
181b-
1, miR-196-1 prec, miR-93-1, miR-223, miR-16-1, miR-10l-1, miR-124a-1, miR-26a-
1, miR-214, miR-27a, miR-24-1, miR-106a, miR-199a-1 and combinations thereof.
In
a related embodiment, the solid cancer is prostate cancer and the miR gene
product is
not miR-15a or miR-16-1. In another embodiment, the solid cancer is prostate
cancer
and the miR gene product is not miR 159-1 or miR-192. In an additional
embodiment
the solid cancer is prostate cancer and the miR gene product is not miR-186,
miR-101-
1, miR-194, miR-215, miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-
182s, miR-1.82as, miR-183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b,
rniR-24-1,
miR-27b, rniR-32, miR-159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-
2,
miR-148b, miR-190, miR-21, miR-301, miR-142s, miR-142as, miR-105-1, or miR-
175. In a further embodiment, the solid cancer is prostate cancer and the miR
gene
product is not miR-21, miR-301, miR-142as, miR-142s, miR-194, miR-215, or miR-
32.
In another embodiment, the solid cancer is prostate cancer and the miR gene
product is
not miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, rniR-
129-1, .miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another
embodiment, the solid cancer is prostate cancer and the miR gene product is
not miR-
181b, miR-181c, miR-181d, miR-30, miR-15b, miR-16-2, miR-153-1, miR-217, miR-
205, miR-204, miR-103, miR-107, miR-129-2, miR-9 or miR-137.
In yet another embodiment, the solid cancer is stomach cancer and the miR gene
product in the test sample is selected from the group consisting of miR-223,
miR-21,
49
=

CA 02811486 2013-03-26
miR-218-2, miR-103-2, miR-92-2, miR-25, miR-136, miR-191, miR-221, miR-125b-2,
miR-103-1, miR-214, miR-222, miR-212 prec, miR-125b-1, miR-100, miR-107, miR-
92-1, miR-96, miR-192, miR-23a, miR-215, miR-7-2, miR-138-2, miR-24-1, miR-
99b,
miR-33b, miR-24-2 and combinations thereof. In a related embodiment, the solid
cancer is stomach cancer and the miR gene product is not miR-15a or miR-16-1.
In
another embodiment, the solid cancer is stomach cancer and the miR gene
product is
not miR 159-1 or miR-192. In an additional embodiment, the solid cancer is
stomach
cancer and the miR gene product is not miR-186, naiR-101-1, miR-194, miR-215,
miR-
106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182as, miR-183,
miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, rniR-24-1, miR-27b, miR-32,
miR-159-
1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190, miR-
21,
miR-301, miR-142s, miR-142as, miR-105-1, or miR-175. In a further embodiment,
the
solid cancer is stomach cancer and the miR gene product is not miR-21, miR-
301, miR-
142as, miR-142s, miR-194, miR_-215, or miR-32. In another embodiment, the
solid
cancer is stomach cancer and the miR gene product is not miR-148, miR-10a, miR-
196-
1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1, miR-153-2, miR-202, miR-
139, let-7a, let-7f, or let-7d. In yet another embodiment, the solid cancer is
stomach
cancer and the miR gene product is not miR-18 lb, miR-181c, miR-181d, miR-30,
miR-
15b, miR-16-2, miR-153-1, miR-217, miR-205, miR-204, miR-103, miR-107, miR-
129-2, miR-9 or miR-137.
The level of at least one miR gene product can be measured in a biological
sample (e.g., cells, tissues) obtained from the subject. For example, a tissue
sample
(e.g., from a tumor) can be removed from a subject suspected of having a solid
cancer
by conventional biopsy techniques. In another embodiment, a blood sample can
be
removed from the subject, and blood cells (e.g., white blood cells) can be
isolated for
DNA extraction by standard techniques. The blood or tissue sample is
preferably
obtained from the subject prior to initiation of radiotherapy, chemotherapy or
other
therapeutic treatment. A corresponding control tissue or blood sample can be
obtained
from unaffected tissues of the subject, from a normal human individual or
population of
normal individuals, or from cultured cells corresponding to the majority of
cells in the =
= subject's sample. The control tissue or blood sample is then processed
along with the
sample from the subject, so that the levels of miR gene product produced from
a given

CA 02811486 2013-03-26
miR gene in cells from the subject's sample can be compared to the
corresponding miR
gene product levels from cells of the control sample. A reference miR
expressien
standard for the biological sample can also be used as a control.
An alteration (e.g., an increase or decrease) in the level of a miR gene
product
previously obtained for a population of normal human controls.
The level of a miR gene product in a sample can be measured using any
51

CA 02811486 2013-03-26
separated by gel electrophoresis on agarose gels according to standard
techniques, and
transferred to nitrocellulose filters. The RNA is then immobilized on the
filters by
heating. Detection and quantification of specific RNA is accomplished using
appropriately labeled DNA or RNA probes complementary to the RNA in question.
See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,
eds.,
2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7.
Suitable probes for Northern blot hybridization of a given miR gene product
can
be produced from the nucleic acid sequences provided in Table la and Table lb
and
, include, but are not limited to, probes having at least about 70%, 75%, 80%,
85%, 90%,
95%, 98%, 99% or complete complementarily to a miR gene product of interest.
Methods for preparation of labeled DNA and RNA probes, and the conditions for
hybridization thereof to target nucleotide sequences, are described in
Molecular
Cloning: A Laboratory Manual, J. Sambrook et at,'eds., 2nd edition, Cold
Spring
Harbor Laboratory Press, 1989, Chapters 10 and 11.
For example, the nucleic acid probe can be labeled with, e.g., a radionuclide,
such as 3H, 32P, 33P, 14C, or 35S; a heavy metal; a ligand capable of
functioning as a
specific binding pair member for a labeled ligand (e.g., biotin, avidin or an
antibody); a
fluorescent molecule; a chemiluminescent molecule; an enzyme or the like.
Probes can be labeled to high specific activity by either the nick translation
method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 or by the random
priming
method of Fienberg et al. (1983), Anal. Biochem. 132:
6 - 13 . The latter is the method of choice for
synthesizing 32P-labeled probes of high specific activity from single-stranded
DNA or
from RNA templates. For example, by replacing preexisting nucleotides with
highly
radioactive nucleotides according to the nick translation method, it is
possible to
prepare 32P-labeled nucleic acid probes with a specific activity well in
excess of 108
cpm/microgram. Autoradiographic detection of hybridization can then be
performed
by exposing hybridized filters to photographic film. Densitometric scanning of
the
photographic films exposed by the hybridized filters provides an accurate
measurement
of miR gene transcript levels. Using another approach, miR gene transcript
levels can
52
=

CA 02811486 2013-03-26
be quantified by computerized imaging systems, such as the Molecular Dynamics
400-
B 2D Phosphorimager available from Atnersham BiosCiences, Piscataway, NJ.
Where radionuclide labeling of DNA or RNA probes is not practical, the
random-primer method can be used to incorporate an analogue, for example, the
dTTP
analogue 5-(N-(N-biotinyl-epsilon-aminocaproy1)-3-aminoally0deoxyuridine
triphosphate, into the probe molecule. The biotinylated probe oligonucleotide
can be
detected by reaction with biotin-binding proteins, such as avidin,
streptavidin, and
. antibodies (e.g., anti-biotin antibodies) coupled to fluorescent dyes or
enzymes that
produce color reactions.
In addition to Northern and other RNA hybridization techniques, determining
the levels of RNA transcripts can be accomplished using the technique of in
situ
hybridization. This technique requires fewer cells than the Northern blotting
technique,
and involves depositing whole cells onto a microscope cover slip and probing
the
nucleic acid content of the cell with a solution containing radioactive or
otherwise
labeled nucleic acid (e.g., cDNA or RNA) probes. This technique is
particularly well-
suited for analyzing tissue biopsy samples from subjects. The practice of the
in situ
hybridization technique is described in more detail in U.S. Pat. No.
5,427,916. Suitable ,
probes for in situ hybridization of a given miR gene product can be produced
from the
nucleic acid sequences provided in Table la and Table lb, and include, but are
not
limited to, probes having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%,
99%
or complete complementarity to a miR gene product of interest, as described
above.
The relative number of miR gene transcripts in cells can also be determined by
reverse transcription of miR gene transcripts, followed by amplification of
the reverse-
transcribed transcripts by polymerase chain reaction (RT-PCR). The levels of
miR
gene transcripts can be quantified in comparison with an internal standard,
for example,
the level of mRNA from a "housekeeping" gene present in the same sample. A
suitable
"housekeeping" gene for use as an internal standard includes, e.g., myosin or
= - glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods for
performing
quantitative and semi-quantitative RT-PCR, and variations thereof, are well
known to
those of skill in the art.
53

CA 02811486 2013-03-26
_
In some instances, it may be desirable to simultaneously determine the
expression level of a plurality of different miR gene products in a sample. In
other
instances, it may be desirable to determine the expression level of the
transcripts of all
known miR genes correlated with a cancer. Assessing cancer-specific expression
levels
= 5 for hundreds of miR genes or gene products is time consuming and
requires a large
amount of total RNA (e.g., at least 20 p.g for each Northern blot) and
autoradiographic
techniques that require radioactive isotopes.
To overcome these limitations, an oligolibrary, in microchip format (i.e., a
microarray), may be constructed containing a set of oligonucleotide (e.g.,
oligodeoxynucleotides) probes that are specific for a set of miR genes. Using
such a
microarray, the expression level of multiple microRNAs in a biological sample
can be
determined by reverse transcribing the RNAs to generate a set of target
oligodeoxynucleotides, and hybridizing them to probe the oligonucleotides on
the
microarray to generate a hybridization, or expression, profile. The
hybridization profile
of the test sample can then be compared to that of a control sample to
determine which
microRNAs have an altered expression level in solid cancer cells. As used
herein,
"probe oligonucleotide" or "probe oligodeoxynucleotide" refers to an
oligonucleotide
that is capable of hybridizing to a target oligonucleotide. "Target
oligonucleotide" or
= "target oligodeoxynucleotide" refers to a molecule to be detected (e.g.,
via
hybridization). By "miR-specific probe oligonucleotide" or "probe
oligonucleotide
specific for a miR" is meant a probe oligonucleotide that has a sequence
selected to
hybridize to a specific miR gene product, or to a reverse transcript of the
specific miR
gene product.
An "expression profile" or "hybridization profile" of a particular sample is .
essentially a fingerprint of the state of the sample; while two states may
have any
particular gene similarly expressed, the evaluation of a number of genes
simultaneously
allows the generation of a gene expression profile that is unique to the state
of the cell. =
That is, normal tissue may be distinguished from cancerous (e.g., tumor)
tissue, and
within cancerous tissue, different prognosis states (for example, good or poor
long term
survival prospects) may be determined. By comparing expression profiles of
solid
cancer tissue in different states, information regarding which genes are
important
(including both up- and down-regulation of genes) in each of these states is
obtained.
=
54
=

CA 02811486 2013-03-26
The identification of sequences that are differentially expressed in solid
cancer tissue,
as well as differential expression resulting in different prognostic outcomes,
allows the
use of this information in a number of ways. For example, a particular
treatment
regime may be evaluated (e.g., to determine whether a chemotherapeutic drug
acts to
improve the long-term prognosis in a particular patient). Similarly, diagnosis
may be
done or confirmed by comparing patient samples with known expression profiles.
Furthermore, these gene expression profiles (or individual genes) allow
screening of
drug candidates that suppress the solid cancer expression profile or convert a
poor
= prognosis profile to a better prognosis profile.
Accordingly, the invention provides methods of diagnosing whether a subject
has, or is at risk for developing, a solid cancer, comprising reverse
transcribing RNA
from a test sample obtained from the subject to provide a set of target
oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to a
microarray
comprising miRNA-specific probe oligonucleotides to provide a hybridization
profile
for the test sample, and comparing the test sample hybridization profile to a
hybridization profile generated from a control sample or reference standard,
wherein an
alteration in the signal of at least one miRNA is indicative of the subject
either having,
or being at risk for developing, a solid cancer. In one embodiment, the
microarray
=
comprises miRNA-specific probe oligonucleotides for a substantial portion of
all
known human miRNAs. In a particular embodiment, the microarray comprises
miRNA-specific probe oligonucleotides for one or more miRNAs selected=from the
group consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b,
miR-199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-
107, miR-32, miR-214, miR-30c, miR-25, milt-221, miR-106a and
combinations thereof.
The microarray can be prepared from gene-specific oligonucleotide probes
generated from known miRNA sequences. The array may contain two different
oligonucleotide probes for each miRNA, one containing the active, mature
sequence
and the other being specific for the precursor of the miRNA. The array may
also
contain controls, such as one or more mouse sequences differing from human
orthologs
by only a few bases, which can serve as controls for hybridization stringency
conditions. tRNAs or other RNAs (e.g., rRNAs, mRNAs) from both species may
also

CA 02811486 2013-03-26
=
be printed on the microchip, providing an internal, relatively stable,
positive control for
specific hybridization. One or more appropriate controls for non-specific
hybridization
may also be included on. the microchip. For this purpose, sequences are
selected based
upon the absence of any homology with any known miRNAs.
The microarray may be fabricated using techniques known in the art. For
example, probe oligonucleotides of an appropriate length, e.g., 40
nucleotides, are 5'-
amine modified at position C6 and printed using commercially available
microarray
systems, e.g., the GeneMachine OniniGridThf 100 Microarrayer and Amersham
CcideLinkm4 activated slides. Labeled cDNA oligomer corresponding to the
target
RNAs is prepared by re-Verse transcribing the target RNA with labeled primer.
Following first strand synthesis, the RNA/DNA hybrids are denatured to degrade
the
RNA templates. The labeled target cDNAs thus prepared are then hybridized to
the
microarray chip under hybridizing conditions, e.g., 6X SSPE/30% formarnide at
25 C
for 18 hours, followed by washing in 0.75X TNT (Tris HC1/NaC1/TweenTm 20) at
37 C
for 40 minutes. At positions on the array where the immobilized probe DNA
recognizes a complementary target cDNA in the sample, hybridization. occurs.
The
labeled target cDNA marks the exact position on the array where binding
occurs,
allowing automatic detection and quantification. The output consists of a list
of
hybridization events, indicating the relative abundance of specific cDNA
sequences,
and therefore the relative abundance of the corresponding complementary miRs,
in the
patient sample. According to one embodiment, the labeled cDNA oligomer is a
biotin-
labeled cDNA, prepared from a biotin-labeled'primer. The microarray is then
processed by direct detection of the biotin-containing transcripts using,
e.g.,
Streptavidin-A1exa647 conjugate, and scanned utilizing conventional scanning
methods. Image intensities of each spot On the array are proportional to the
abundance
of the corresponding miR in the patient sample.
The use of the array has several advantages for miRNA expression detection.
First, the global expression of several hundred genes can be identified in the
same
sample at one time point Second, through careful design of the oligonucleotide
probes,
expression of both mature and precursor molecules (*Inn be identified. Third,
in
comparison with Northern blot analysis, the chip requires a small amount of
RNA, and
provides reproducible results using 2.5pg of total RNA. The relatively limited
number
56

CA 02811486 2013-03-26
of miRNAs (a few hundred per species) allows the construction of a common
microarray for several species, with distinct oligonucleotide probes for each.
Such a
tool would allow for analysis of trans-species expression for each known miR
under
various conditions:
In addition to use for quantitative expression level assays of specific miRs,
a
microchip containing miRNA-specific probe oligonucleotides corresponding to a
substantial portion of the miRNome, preferably the entire miRNorne, may be
employed
to carry out miR gene expression profiling, for analysis of miR expression
patterns_
Distinct miR signatures can be associated with established disease markers, or
directly
with a disease state.
According to the expression profiling methods described herein, total RNA
from a sample from a subject suspected of having a cancer (e.g., a solid
cancer) is
quantitatively reverse transcribed to provide a set of labeled target
oligodeoxynucleotides complementary to the RNA in the sample. The target
oligodeoxynucleotides are then hybridized to a microarray comprising miRNA-
specific
probe oligonucleotides to provide a hybridization profile for the sample. The
result is a
hybridization profile for the sample representing the expression pattern of
miRNA in
the sample. The hybridization profile comprises the signal from the binding of
the
target oligodeoxynucleotides from the sample to the miRNA-specific probe
oligonucleotides in the microarray. The profile may be recorded as the
presence or
absence of binding (signal vs. zero signal). More preferably, the profile
recorded
includes the intensity of the signal from each hybridization. The profile is
compared to
the hybridization profile generated from a normal, Le., noncancerous, control
sample.
An alteration in the signal is indicative of the presence of, or propensity to
develop,
cancer in the subject.
Other techniques for measuring miR gene expression are also within the skill
in
the art, and include various techniques for measuring rates of RNA
transcription and
degradation.
The invention also provides methods of determining the prognosis of a subject
with a solid cancer, comprising measuring the level of at least one miR gene
product,
which is associated with a particular prognosis in a solid cancer (e.g., a
good or positive
prognosis, a poor or adverse prognosis), in a test sample from the subject.
According to
=
57 =

CA 02811486 2013-03-26
these methods, an alteration in the level of a miR gene product that is
associated with a
particular prognosis in the test sample, as compared to the level of a
corresponding miR
gene product in a control sample, is indicative of the subject having a solid
cancer with
a particular prognosis. In one embodiment, the miR gene product is associated
with an
adverse (i.e., poor) prognosis. Examples of an adverse prognosis include, but
are not
limited to, low survival rate and rapid disease progression. In certain
embodiments, the
level of the at least one miR gene product is measured by reverse transcribing
RNA
from a test sample obtained from the subject to provide a set of target
oligodeoxynueleotides, hybridizing the target oligodeoxynucleotides to a
micioarray
that comprises niiRNA-specific probe oligonucleotides to provide a
hybridization
profile for the test sample, and comparing the test sample hybridization
profile to a
hybridization profile generated from a control sample.
Without wishing to be bound by any one theory, it is believed that alterations
in
the level of one or more miR gene products in cells can result in the
deregulation of one
or more intended targets for these miRs, which can lead to the formation of
solid
cancers. Therefore, altering the level of the miR gene product (e.g., by
decreasing the
level of a miR gene product that is up-regulated in solid cancer cells, by
increasing the
level of a miR gene product that is down-regulated in solid cancer cells) may
successfully treat the solid cancer.
Accordingly, the present invention encompasses methods of inhibiting
tumorigenesis in a subject who has, or is suspected of having, a solid cancer
wherein at
least one miR gene product is deregulated (e.g., down-regulated, up-regulated)
in the
cancer cells of the subject. When the at least one isolated miR gene product
is down-
regulated in the cancer cells (e.g., miR-145, miR-155, miR-218-2), the method
comprises administering an effective amount of the at least one isolated miR
gene
product, or an isolated variant or biologically-active fragment thereof, such
that
proliferation of cancer cells in the subject is inhibited. In one embodiment,
the isolated
miR gene product that is administered is not miR-1 5a or miR-16-.1. In another
embodiment, the miR gene product is not miR 159-1 or miR-192. In an additional
embodiment, the miR gene product is not miR-186, miR-101-1, miR-194, miR-215,
miR-106b, miR-25, miR-93, miR-29b, miR-29a, miR-96, miR-182s, miR-182as, miR-
183, miR-129-1, let-7a-1, let-7d, let-7f-1, miR-23b, miR-24-1, miR-27b, miR-
32, miR-
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CA 02811486 2013-03-26
159-1, miR-192, miR-125b-1, let-7a-2, miR-100, miR-196-2, miR-148b, miR-190,
miR-21, miR-301, miR-142s, miR-142as, rniR-105-1, or miR-175. In a further
embodiment, the miR gene product is not miR-21, miR-301, miR-142as, miR-142s,
miR-194, miR-215, or miR-32. In another embodiment, the miR gene product is
not
miR-148, miR-10a, miR-196-1, miR-152, miR-196-2, miR-148b, miR-10b, miR-129-1,
miR-153-2, miR-202, miR-139, let-7a, let-7f, or let-7d. In yet another
embodiment, the
miR gene product is not miR-30, miR-15b, miR-16-2, miR-217, miR-205, miR-204,
miR-103, miR-107, miR-9, and miR-137. In a further embodiment, the miR gene
product is not miR-145, miR-21, miR-155, miR-10b, miR-125b-1, miR-125b-2,
let7a-
2, let7a-3, let-7d, rniR-122a, miR-191, miR-206, miR-210, let-7i, miR-009-1
(miR131-
1), miR-34 (miR-170), miR-102 (miR-29b), miR-123 (miR-126), miR-140-as, miR-
125a, miR-194, miR-204, miR-213, let-7f-2, miR-101, miR-128b, miR-136, miR-
143,
miR-149, miR-191, miR-196-1, miR-196-2, miR-202, miR-103-1, or miR-30c. In
another embodiment, the miR gene product is not miR-21, miR-125b-1, let-7a-2,
let-7i,
miR-100, let-7g, miR-31, miR-32a-1, miR-33b, miR-34a-2, miR-101-1, miR-135-1,
miR-142as, miR-142s, miR-144, miR-301, miR-29c, miR-30c, miR-106a, or miR-29b-
1.
For example, when a miR gene product is down-regulated in a cancer cell in a
subject, administering an effective amount of an isolated miR gene product to
the
subject can inhibit proliferation of the cancer cell. The isolated miR gene
product that
is administered to the subject can be identical to the endogenous wild-type
miR gene
product (e.g., a miR_ gene product shown in Table la or Table lb) that is down
regulated in the cancer cell or it can be a variant or biologically-active
fragment
thereof. As defined herein, a "variant" of a miR gene product refers to a
miRNA that
has less than 100% identity to a corresponding wild-type miR gene product and
possesses one or more biological activities of the corresponding wild-type miR
gene
product. Examples of such biological activities include, but are not limited
to,
inhibition of expression of a target RNA molecule (e.g., inhibiting
translation of a
target RNA molecule, modulating the stability of a target RNA molecule,
inhibiting
processing of a target RNA molecule) and inhibition of a cellular process
associated
with solid cancer (e.g., cell differentiation, tell growth, cell death). These
variants
include species variants and variants that are the consequence Of one or more
mutations
59

CA 02811486 2013-03-26
(e.g., a substitution, a deletion, an insertion) in a miR gene_ In certain
embodiments,
the variant is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%
identical to
a corresponding wild-type miR gene product.
As defined herein, a "biologically-active fragment" of a miR gene product
refers
to an RNA fragment of a miR gene product that possesses one or more biological
activities of a corresponding wild-type miR gene product. As described above,
, examples of such biological activities include, but are not limited to,
inhibition of
expression of a target RNA molecule and inhibition of a cellular process
associated
with solid cancer, In certain embodiments, the biologically-active fragment is
at least
about 5, 7, 10, 12, 15, or 17 nucleotides in length. In a particular
embodiment, an
isolated miR gene product can be administered to a subject in combination with
one or
more additional anti-cancer treatments. Suitable anti-cancer treatments
include, but are
not limited to, chemotherapy, radiation therapy and combinations thereof
(e.g.,
chemoradiation).
When the at least one isolated miR gene product is up-regulated in the cancer
cells, the method comprises administering to the subject an effective amount
of at least
one compound for inhibiting expression of the at least one miR gene product,
referred
to herein as miR gene expression-inhibition compounds, such that proliferation
of solid
cancer cells is inhibited. In a particular embodiment, the at least one miR
expression-
inhibition compound is specific for a miR gene product selected from the group
consisting of miR-21, miR-17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-
199a-1, miR-24-1, miR-24-2, miR-146, miR-155, miR-181b-1, miR-20a, miR-107,
miR-32, miR-92-2, miR-214, miR-30c, miR-25, miR-221, miR-106a and combinations
thereof. A miR gene expression-inhibiting compound can be administered to a
subject
in combination with one or more additional anti-cancer treatments. Suitable
anti-cancer
treatments include, but are not limited to, chemotherapy, radiation therapy
and
combinations thereof (e.g., chemoradiation).
The terms "treat", "treating" and "treatment", as used herein, refer to
ameliorating symptoms associated with a disease or condition, for example, a
solid
cancer, including preventing or delaying the onset of the disease symptoms,
and/or
= lessening the severity or frequency of symptoms of the disease or
condition. The terms
"subject", "patient" and "individual" are defined herein to include animals,
such as

CA 02811486 2013-03-26
mammals, including, but not limited to, primates, cows, sheep, goats, horses,
dogs, cats,
rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine,
feline, rodent, or
murine species. In a preferred embodiment, the animal is a human.
As used herein, an "effective amount" of an isolated miR gene product is an
amount sufficient to inhibit proliferation of a cancer cell in a subject
suffering from a
solid cancer. One skilled in the art can readily determine an effective amount
of a miR =
gene product to be administered to a given subject, by taking into account
factors, such as
the size and weight of the subject; the extent of disease penetration; the
age, health and sex
of the subject; the route of administration; and whether the administration is
regional or
systemic.
For example, an effective amount of an isolated miR gene product can be based
on the approximate weight of a tumor mass to be treated. The approximate
weight of a
tumor mass can be determined by calculating the approximate volume of the
mass,
wherein one cubic centimeter of volume is roughly equivalent to one gram. An
effective amount of the isolated miR gene product based on the weight of a
tumor mass
can be in the range of about 10-500 micrograms/gram of tumor mass. In certain
embodiments, the tumor mass can be at least about 10 micrograms/gram of tumor
mass,
at least about 60 micrograms/gram of tumor mass or at least about 100
micrograms/gram of tumor mass.
An effective amount of an isolated miR gene product can also be based on the
approximate or estimated body weight of a subject to be treated. Preferably,
such
effective amounts are administered parenterally or enterally, as described
herein. For
example, an effective amount of the isolated miR gene product is administered
to a
subject can range from about 5 3000 micrograms/kg of body weight, from about
700 -
1000 micrograms/kg of body weight, or greater than about 1000 micrograms/kg of
body weight.
One skilled in the art can also readily determine an appropriate dosage
regimen
for the administration of an isolated miR gene product to a given subject. For
example,
a miR gene product can be administered to the subject once (e.g., as a single
injection
or deposition). Alternatively, a miR gene product can be administered once or
twice
daily to a subject for a period of from about three to about twenty-eight
days, more
particularly from about seven to about ten days. In a particular dosage
regimen, a miR
61

CA 02811486 2013-03-26
gene product is administered once a day for seven days. Where a dosage regimen
comprises multiple administrations, it is understood that the effective amount
of the
miR gene product administered to the subject can comprise the total amount of
gene
product administered over the entire dosage regimen.
As used herein, an "isolated" miR gene product is one that is synthesized, or
altered or removed from the natural state through human intervention. For
example, a
synthetic miR gene product, or a miR gene product partially or completely
separated
from the coexisting materials of its natural state, is considered to be
"isolated." An
isolated miR gene product can exist in substantially-purified form, or can
exist in a cell
into which the miR gene product has been delivered. Thus, a miR gene product
that is
deliberately delivered to, or expressed in, a cell is considered an "isolated"
miR gene
product. A miR gene product produced inside a cell from a miR precursor
molecule is
also considered to be an "isolated" molecule. According to the invention, the
isolated
miR gene products described herein can be used for the manufacture of a
medicament
for treating a solid cancer in a subject (e.g., a human).
Isolated miR gene products can be obtained using a number of standard
techniques. For example, the miR gene products can be chemically synthesized
or
recombinantly produced using methods known in the art. In one embodiment, miR
gene products are chemically synthesized using appropriately protected
ribonucleoside
phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers
of
synthetic RNA molecules or synthesis reagents include, e.g., Froligo (Hamburg,
Germany), Dharmacon Research (Lafayette, CO, U.S.A.), Pierce Chemical (part of
Perbio Science, Rockford, IL, U.S.A.), Glen Research (Sterling, VA, U.S.A.),
ChemGenes (Ashland, MA, U.S.A.) and Cruachem (Glasgow, UK).
Alternatively, the miR gene products can be expressed from recombinant
circular or linear DNA plasmids using any suitable promoter. Suitable
promoters for
expressing RNA from a plasmid include, e.g., the U6 or H1 RNA pol III promoter
sequences, or the cytomegalovirus promoters. Selection of other suitable
promoters is
within the skill in the art. The recombinant plasmids of the invention can
also comprise
inducible or regulatable promoters for expression of the miR gene products in
cancer
cells.
62

CA 02811486 2013-03-26
The miR gene products that are expressed from recombinant plasmids can be
isolated from cultured cell expression systems by standard techniques. The miR
gene
products that are expressed from recombinant plasmids can also be delivered
to, and
expressed directly in, the cancer cells. The use of recombinant plasmids to
deliver the
miR gene products to cancer cells is discussed in more detail below.
The miR gene products can be expressed from a separate recombinant plasmid,
or they can be expressed from the same recombinant plasmid. In one embodiment,
the
miR gene products are expressed as RNA precursor molecules from a single
plasmid,
and the precursor molecules are processed into the functional miR gene product
by a
suitable processing system, including, but not limited to, processing systems
extant
within a cancer cell. Other suitable processing systems include, e.g., the in
vitro
Drosophila cell lysate system (e.g., as described in U.S. Published Patent
Application
No. 2002/0086356 to Tuschl et al.) and the E. Coil RNAse DJ system (e.g., as
described in
U.S. Published Patent Application No. 2004/0014113 to Yang et al.).
Selection of plasmids suitable for expressing the miR gene products, methods
for inserting nucleic acid sequences into the plasmid to express the gene
products, and
methods of delivering the recombinant plasmid to the cells of interest are
within the
skill in the art. See, for example, Zeng et al. (2002), Molecular Cell 9:1327-
1333;
Tuschl (2002), Nat. Biotechnol, 20:446-448; Brtunmelkamp et al. (2002),
Science
296:550-553; Miyagishi etal. (2002), Nat. Blotechnot 20:497-500; Paddison et
al.
(2002), Genes Dev. 16:948-958; Lee etal. (2002), Nat. Biotechnol. 20:500-505;
and
Paul etal. (2002), Nat. Biotechnol. 20:505-508.
In one embodiment, a plasmid expressing the miR gene products comprises a
sequence encoding a miR precursor RNA under the control of the CMV
intermediate-
early promoter. As used herein, "under the control" of a promoter means that
the
= nucleic acid sequences encoding the miR gene product are located 3' of
the promoter,
so that the promoter can initiate transcription of the miR gene product coding
sequences.
63

CA 02811486 2013-03-26
The miR gene products can also be expressed from recombinant viral vectors.
It is contemplated that the miR gene products can be expressed from two
separate
recombinant viral vectors, or from the same viral vector. The RNA expressed
from the
recombinant viral vectors can either be isolated from cultured cell expression
systems
by standard techniques, or can be expressed directly in cancer cells. The use
of
recombinant viral vectors to deliver the miR gene products to cancer cells is
discussed
in more detail below.
The recombinant viral vectors of the invention comprise sequences encoding the
miR gene products and any suitable promoter for expressing the RNA sequences.
Suitable promoters include, but are not limited to, the U6 or Hi RNA pol III
promoter
sequences, or the cytomegalovirus promoters. Selection of other suitable
promoters is
within the skill in the art. The recombinant viral vectors of the invention
can also
comprise inducible or regulatable promoters for expression of the miR gene
products in
a cancer cell.
Any viral vector capable of accepting the coding sequences for the miR gene
products can be used; for example, vectors derived from adenovirus (AV); adeno-
associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses,
murine
leukemia virus); herpes virus, and the like. The tropism of the viral vectors
can be
modified by pseudotyping the vectors with envelope proteins or other surface
antigens
from other viruses, or by substituting different viral capsid proteins, as
appropriate.
For example, lentiviral vectors of the invention can be pseudotyped with
surface
proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the
like.
AAV vectors of the invention can be made to target different cells by
engineering the
vectors to express diffeient capsid protein serotypes. For example, an AAV
vector
expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This
serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5
capsid
gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors
that
express different capsid protein serotypes are within the skill in the art;
see, e.g.,
Rabinowitz, J.E., et al. (2002), Virol. 76:791-801.
5
- 30
Selection of recombinant viral vectors suitable for use in the invention,
methods
for inserting nucleic acid sequences for expressing RNA into the vector,
methods of
64

CA 02811486 2013-03-26
= delivering the viral vector to the cells of interest, and recovery of the
expressed RNA
products are within the skill in the art. See, for example, Domburg (1995),
Gene
Therapy 2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990),
Hum.
Gene Therapy 1:5-14; and Anderson (1998), Nature 392:25-30.
Particularly suitable viral vectors are those derived from AV and AAV. A
suitable ,
AV vector for expressing the miR gene products, a method for constructing the
recombinant
AV vector, and a method for delivering the vector into target cells, are
described in Xia et al.
(2002), Nat. Biotech. 20:1006-1010. Suitable AAV vectors for expressing the
miR gene
=
products, methods for constructing the recombinant AAV vector, and methods for
delivering
.
the vectors into target cells are described in Samulski et al. (1987),J. ViroL
61:3096-3101;
Fisher etal. (1996), J Virol, 70:520-532; Samulski et al. (1989), J ViroL
63:3822-3826;
U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent
Application No. WO
94/13788; and International Patent Application No. WO 93/24641. In one
embodiment, the
miR gene products are expressed from a single recombinant AAV vector
comprising the
CMV intermediate early promoter.
In a certain embodiment, a recombinant AAV viral vector of the invention
comprises a nucleic acid sequence encoding a miR precursor RNA in operable
connection with a polyT termination sequence under the control of a human U6
RNA
promoter_ As used herein, "in operable connection with a polyT termination
sequence"
= means that the nucleic acid sequences encoding the sense or antisense
strands are
immediately adjacent to the polyT termination signal in the 5' direction.
During
transcription of the miR sequences from the vector, the polyT termination
signals act to
terminate transcription. =
In other embodiments of the treatment methods of the invention, an effective
amount of at least one compound that inhibits miR expression can be
administered to
the subject. As used herein, "inhibiting miR expression" means that the
production of
the precursor and/or active, mature form of miR gene product after treatment
is less
than the amount produced prior to treatment. One skilled in the .art can
readily
determine whether miR expression has been inhibited in a cancer cell, using,
for

CA 02811486 2013-03-26
example, the techniques for determining miR transcript level discussed above
for the
diagnostic method. Inhibition can occur at the level of gene expression (i.e.,
by
inhibiting transcription of a miR gene encoding the miR gene product) or at
the level of
processing (e.g., by inhibiting processing of a miR precursor into a mature,
active miR).
As used herein, an "effective amount" of a compound that inhibits miR
expression is an amount sufficient to inhibit proliferation of a cancer cell
in a subject
suffering from a cancer (e.g., a solid cancer). One skilled in the art can
readily
determine an effective amount of a miR expression-inhibition compound to be
administered to a given subject, by taking into account factors, such as the
size and weight
of the subject; the extent of disease penetration; the age, health and sex of
the subject; the
route of administration; and whether the administration is regional or
systemic.
For example, an effective amount of the expression-inhibition compound can be
based on the approximate weight of a tumor mass to be treated, as described
herein. An
effective amount of a compound that inhibits miR expression can also be based
on the
approximate or estimated body weight of a subject to be treated, as described
herein.
One skilled in the art can also readily determine an appropriate dosage
regimen
for administering a compound that inhibits miR expression to a given subject.
Suitable compounds for inhibiting miR gene expression include double-stranded
RNA (such as short- or small-interfering RNA or "siRNA"), antisense nucleic
acids,
and enzymatic RNA molecules, such as ribozymes. Each of these compounds can be
=
targeted to a given miR gene product and interfere with the expression of
(e.g., inhibit
translation of, induce cleavage or destruction of) the target miR gene
product.
For example, expression of a given miR gene can be inhibited by inducing RNA
interference of the miR gene with an isolated double-stranded RNA ("dsRNA")
molecule which has at least 90%, for example at least 95%, at least 98%, at
least 99%,
or 100%, sequence homology with at least a portion of the miR gene product. In
a
particular embodiment, the dsRNA molecule is a "short or small interfering
RNA" or
"siRNA."
siRNA useful in the present methods comprise short double-stranded RNA from
about 17 nucleotides to about 29 nucleotides in length, preferably from about
19 to
about 25 nucleotides in length. The siRNA comprise a sense RNA strand and a
complementary antisense RNA strand annealed together by standard Watson-Crick
=
66

CA 02811486 2013-03-26
base-pairing interactions (hereinafter "base-paired"). The sense strand
comprises a
nucleic acid sequence that is substantially identical to a nucleic acid
sequence contained
within the target miR gene product.
As used herein, a nucleic acid sequence in an siRNA which is "substantially
molecule in which two complementary portions are base-paired and are
covalently
The siRNA can also be altered RNA that differs from naturally-occurring RNA
by the addition, deletion, substitution and/or alteration of one or more
nucleotides. Such
alterations can include addition of non-nucleotide material, such as to the
end(s) of the
siRNA or to one or more internal nucleotides of the siRNA, or modifications
that make
One or both strands of the siRNA can also comprise a 3' overhang. As used
herein, a "3' overhang" refers to at least one unpaired nucleotide extending
from the 3'-
end of a duplexed RNA strand. Thus, in certain embodiments, the siRNA
comprises at
The siRNA can be produced chemically or biologically, or can be expressed
from a recombinant plasmid or viral vector, as described above for the
isolated miR
gene products. Exemplary methods for producing and testing dsRNA or siRNA
molecules are described in U.S. Published Patent Application No. 2002/0173478
to
67
=

CA 02811486 2013-03-26
Expression of a given miR gene can also be inhibited by an antisense nucleic
acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid
molecule that
binds to target RNA by means of RNA-RNA, RNA-DNA or RNA-peptide nucleic acid
interactions, which alters the activity of the target RNA. Antisense nucleic
acids
suitable for use in the present methods are single-stranded nucleic acids
(e.g., RNA,
DNA, RNA-DNA chimeras, peptide nucleic acid (PNA)) that generally comprise a
nucleic acid sequence complementary to a contiguous nucleic acid sequence in a
miR
gene product. The antisense nucleic acid can comprise a nucleic acid sequence
that is
50-100% complementary, 75-100% complementary, or 95-100% complementary to a
contiguous nucleic acid sequence in a miR gene product. Nucleic acid sequences
for
the miR gene products are provided in Tables la and lb. Without wishing to be
bound
by any theory, it is believed that the antisense nucleic acids activate RNase
H or
another cellular nuclease that digests the miR gene product/antisense nucleic
acid
duplex.
Antisense nucleic acids can also contain modifications to the nucleic acid
backbone or to the sugar and base moieties (or their equivalent) to enhance
target "
specificity, nuclease resistance, delivery or other properties related to
efficacy of the
molecule_ Such modifications include cholesterol moieties, duplex
intercalators, such
as acridine, or one or more nuclease-resistant groups.
Antisense nucleic acids can be produced chemically or biologically, or can be
expressed from a recombinant plasmid or viral vector, as described above for
the
isolated miR gene products. Exemplary methods for producing and testing are
within
the skill in the art; see, e.g., Stein and Cheng (1993), Science 261:1004 and
U.S. Pat.
No. 5,849,902 to Woolf et al.
Expression of a given miR gene can also be inhibited by an enzymatic nucleic
acid. As used herein, an "enzymatic nucleic acid" refers to a nucleic acid
comprising a
substrate binding region that has complementarity to a contiguous nucleic acid
sequence of a miR gene product, and which is able to specifically cleave the
miR gene
product. The enzymatic nucleic acid substrate binding region can be, for
example, 50-
100% complementary, 75-100% complementary, or 95400% complementary to a
contiguous nucleic acid sequence in a miR gene product. The enzymatic nucleic
acids
68

CA 02811486 2013-03-26
can also comprise modifications at the base, sugar, and/or phosphate groups.
An
exemplary enzymatic nucleic acid for use in the present methods is a ribozyme.
The enzymatic nucleic acids can be produced chemically or biologically, or can
be expressed from a recombinant plasrnid or viral vector, as described above
for the
isolated miR gene products. Exemplary methods for producing and testing dsRNA
or
siRNA molecules are described in Werner and Uhlenbeck (1995), Nucl. Acids Res.
23:2092-96; Hamrnami et al. (1999), Antisense and Nucleic Acid Drug Dev. 9:25-
31;
and U.S. Pat. No. 4,987,071 to Cech et al.
Administration of at least one miR gene product, or at least one compound for
inhibiting miR expression, will inhibit the proliferation of cancer cells in a
subject who
has a solid cancer.. As used herein, to "inhibit the proliferation of a cancer
cell" means
to kill the cell, or permanently or temporarily arrest or slow the growth of
the cell.
Inhibition of cancer cell proliferation can be inferred if the number of such
cells in the
subject remains constant or decreases after administration of the miR gene
products or
miR gene expression-inhibition compounds. An inhibition of cancer cell
proliferation
can also be inferred if the absolute number of such cells increases, but the
rate of tumor
growth decreases.
The number of cancer cells in the body of a subject can be determined by
direct
measurement, or by estimation from the size of primary or metastatic tumor
masses.
For example, the number of cancer cells in a subject can be measured by .
immunohistological methods, flow cytornetry, or other techniques designed to
detect
characteristic surface markers of cancer cells.
The size of a tumor mass can be ascertained by direct visual observation, or
by
= 25 diagnostic imaging methods, such as X-ray, magnetic resonance imaging,
ultrasound,
and scintigraphy. Diagnostic imaging methods used to ascertain size of the
tumor mass
can be employed with or without contrast agents, as is known in the art. The
size of a
tumor mass can also be ascertained by physical means, such as palpation of the
tissue
mass or measurement of the tissue mass with a measuring instrument, such as a
caliper.
The miR gene products or miR gene expression-inhibition compounds can be
administered to a subject by any means suitable for delivering these compounds
to
cancer cells of the subject. For example, the miR gene products or miR
expression-
69

CA 02811486 2013-03-26
inhibition compounds can be administered by methods suitable to transfect
cells of the
subject with these compounds, or with nucleic acids comprising sequences
encoding
these compounds. In one embodiment, the cells are transfected with a plasmid
or viral
vector comprising sequences encoding at least one miR gene product or miR gene
expression-inhibition compound.
Transfection methods for eukaryotic cells are well known in the art, and
include, e.g., direct injection of the nucleic acid into the nucleus or
pronucleus of a cell;
electroporation; liposome transfer or transfer mediated by lipophilic
materials;
receptor-mediated nucleic acid delivery, bioballistic or particle
acceleration; calcium
phosphate precipitation, and tTansfection mediated by viral vectors.
For example, cells can be transfected with a liposomal transfer compound,
e.g.,
DOTAP (N41-(2,3-dioleoyloxy)propyli-N,N,N-trimethyl-ammonium methylsulfate,
Boehringer-Mannheim) or an equivalent, such as LIPOFECT1N. The amount of
nucleic acid used is not critical to the practice of the invention; acceptable
results may
be achieved with 0.1-100 micrograms of nucleic acid/105 cells. For example, a
ratio of
about 0.5 microgram of plasrnid vector in 3 micrograms of DOTAP per 105 cells
can
be used.
A miR gene product or miR gene expression-inhibition compound can also be
administered to a subject by any suitable enteral or parenteral administration
route.
Suitable enteral administration routes for the present methods include, e.g.,
oral, rectal,
or intranasal delivery. Suitable parenteral administration routes include,
e.g.,
intravascular administration (e.g., intravenous bolus injection, intravenous
infusion,
intra-arterial bolus injection, intra-arterial infusion and catheter
instillation into the
vasculature); pen- and intra-tissue injection (e.g., peri-tumoral and intra-
tumoral
injection, intra-retinal injection, or subretinal injection); subcutaneous
injection or
deposition, including subcutaneous infusion (such as by osmotic pumps); direct
application to the tissue of interest, for example by a catheter or other
placement device
(e.g., a retinal pellet or a suppository or an implant comprising a porous,
non-porous, or
gelatinous material); and inhalation. Particularly suitable administration
routes are
injectibn, infusion and direct injection into the tumor.
In the present methods, a miR gene product or miR gene product expression-
inhibition compound can be administered to the subject either as naked RNA,
in.

CA 02811486 2013-03-26
combination with a delivery reagent, or as a nucleic acid (e.g., a recombinant
plasmid
or viral vector) comprising sequences that express the miR gene product or miR
gene
product expression-inhibition compound. Suitable delivery reagents include,
e.g., the
Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin;
polycations
(e.g., polylysine), and liposomes.
Recombinant plasmids and viral vectors comprising sequences that express the
miR gene products or miR gene expression-inhibition compounds, and techniques
for
delivering such plasmids and vectors to cancer cells, are discussed herein
and/or are
well known in the art.
In a particular embodiment, liposomes are used to deliver a miR gene product
or
miR gene expression-inhibition compound (or nucleic acids comprising sequences
encoding them) to a subject. Liposomes can also increase the blood half-life
of the
gene products or nucleic acids. Suitable liposomes for use in the invention
can be
formed from standard vesicle-forming lipids, which generally include neutral
or
negatively charged phospholipids and a sterol, such as cholesterol. The
selection of
lipids is generally guided by consideration of factors, such as the desired
liposome size
and half-life of the liposomes in the blood stream. A variety of methods are
known for
preparing liposomes, for example, as described in Szoka et aL (1980), Ann.
Rev.
Biophys. Bioeng. 9:467; and -U.S. Pat. Nos. 4,235,871, 4,501,728,4,837,028,
and
5,019,369.
The liposomes for use in the present methods can comprise a ligand molecule
that targets the liposome to cancer cells. Ligands that bind to receptors
prevalent in
cancer cells, such as monoclonal antibodies that bind to tumor cell antigens,
are
preferred.
The liposomes for use in the present methods can also be modified so as to
avoid clearance by the mononuclear macrophage system ("MMS") and
reticuloendothelial system ("RES"). Such modified liposomes have opsonization-
inhibition moieties on the surface or incorporated into the liposome
structure. In a
particularly preferred embodiment, a liposome of the invention can comprise
both an
opsonization-inhibition moiety and a ligand.
Opsonization-inhibiting moieties for use in preparing the liposomes of the
invention are typically large hydrophilic polymers that are bound to the
liposome
71

CA 02811486 2013-03-26
Membrane. As used herein, an opsonization-inhibiting moiety is "bound" to a
liposome
membrane when it is chemically or physically attached to the membrane, e.g.,
by the
intercalation of a lipid-soluble anchor into the membrane itself, or by
binding directly
to active groups of membrane lipids. These opsonization-inhibiting hydrophilic
polymers form a protective surface layer that significantly decreases the
uptake of the
liposomes by the MMS and RES; e.g., as described in U.S. Pat. No. 4,920,016.
Opsonizati on-inhibiting moieties suitable for modifying liposomes are
preferably water-soluble polymers with a number-average molecular weight from
about
500 to about 40,000 daltons, and more preferably from about 2,000 to about
20,000
daltons. Such polymers include polyethylene glycol (PEG) or polypropylene
glycol
(PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate;
synthetic
polymers, such as polyacrylarnide or poly N-vinyl pyrrolidone; linear,
branched, or
dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g.,
polyvinylalcohol
and polyxylitol to which carboxylic or amino groups are chemically linked, as
well- as-
gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or
methoxy PPG, or derivatives thereof, are also suitable. In addition, the
opsonization-
inhibiting polymer can be a block copolymer of PEG and either a polyamino
acid,
polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The
opsonization-inhibiting polymers can also be natural polysaccharides
containing amino
acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,
mannuronic acid,
hyaluronic acid, pectic acid, neurarninic acid, alginic acid, carrageenan;
aminated
polysaccharides or oligosaccharides (linear or branched); or carboxylated
polysaccharides or oligosaccharides, e.g., reacted with derivatives of
carbonic acids
with resultant linking of carboxylic groups. Preferably, the opsonization-
inhibiting
moiety is a PEG, PPG, or a derivative thereof. Liposomes modified with PEG or
PEG-
derivatives are sometimes called "PEGylated liposomes."
The opsonization-inhibiting moiety can be bound to the liposome membrane by
any one of numerous well-known techniques. For example, an N-
hydroxysuccinimide
ester of PEG can be bound to a phosphatidyl-ethanolarnine lipid-soluble
anchor, and
then bound to a membrane. Similarly, a dextran polymer can be derivatized with
a
72

CA 02811486 2013-03-26
stearylamine lipid-soluble anchor via reductive amination using Na(CN)B113 and
a
solvent mixture, such as tetrahydrofuran and water in a 30:12 ratio at 60 C.
Liposomes modified with opsonization-inhibition moieties remain in the
circulation much longer than unmodified liposomes. For this reason, such
liposomes
are sometimes called "stealth" liposomes. Stealth liposomes are known to
accumulate
in tissues fed by porous or "leaky" microvasculature. Thus, tissue
characterized by
such microvasculature defects, for example, solid tumors, will efficiently
accumulate
these liposomes; see Gabizon, et al. (1988), Proc. Natl. Mad. Sc., U.S.A.,
18:6949-53.
In addition, the reduced uptake by the RES lowers the toxicity of stealth
liposomes by
preventing significant accumulation of the liposomes in the liver and spleen.
Thus,
liposomes that are modified with opsonization-inhibition moieties are
particularly
suited to deliver the miR gene products or miR gene expression-inhibition
compounds
(or nucleic acids comprising sequences encoding them) to tumor cells.
The miR gene products or miR gene expression-inhibition compounds can be
formulated as pharmaceutical compositions, sometimes called "medicaments,"
prior to
administering them to a subject, according to techniques known in the art.
Accordingly, the invention encompasses pharmaceutical compositions for
treating a
solid cancer. In one embodiment, the pharmaceutical composition comprises at
least
one isolated miR gene product, or an isolated variant or biologically-active
fragment
thereof, and a pharmaceutically-acceptable carrier. In a particular
embodiment, the at
least one miR gene product corresponds to a miR gene product that has a
decreased
level of expression in solid cancer cells relative to suitable control cells.
In certain
embodiments the isolated miR gene product is selected from the group
consisting of
miR-145, miR-155, miR-218-2 combinations thereof.
In other embodiments, the pharmaceutical compositions of the invention
comprise at least one miR expression-inhibition compound. In a particular
embodiment, the at least one miR gene expression-inhibition compound is
specific for a
miR gene whose expression is greater in solid cancer cells than control cells.
In certain
embodiments, the miR gene expression-inhibition compound is specific for one
or more
miR gene products selected from the group consisting of miR-21, miR-17-5p, miR-
191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2, miR-146,
73

CA 02811486 2013-03-26
miR-155, miR-181b-1, miR-20a, miR-107, miR-32, miR-92-2, miR-214, miR-30c,
miR-25, miR-221, miR-106a and combinations thereof.
Pharmaceutical compositions of the present invention are characterized as
being
at least sterile and pyrogen-free. As used herein, "pharmaceutical
compositions"
include formulations for human and veterinary use. Methods for preparing
pharmaceutical compositions of the invention are within the skill in the art,
for example
as described in-Remington's Pharmaceutical Science, 17th ed., Mack Publishing
Company, Easton, Pa. (1985).
The present pharmaceutical compositions comprise at least one miR gene
product or miR gene expression-inhibition compound (or at least one nucleic
acid
comprising sequences encoding them) (e.g., 0.1 to 90% by weight), or a
physiologically-acceptable salt thereof, mixed with a pharmaceutically-
acceptable
carrier. In certain embodiments, the pharmaceutical compositions of the
invention
additionally comprise one or more anti-cancer agents (e.g., chemotherapeutic
agents).The pharmaceutical formulations of the invention can also comprise at
least one
miR gene product or miR gene expression-inhibition compound (or at least one
nucleic
acid comprising sequences encoding them), which are encapsulated by liposomes
and a
pharmaceutically-acceptable carrier. In one embodiment, the pharmaceutical
composition comprises a miR gene or gene product that is not miR-15 and/or miR-
16.
Especially suitable pharmaceutically-acceptable carriers are water, buffered
water, normal saline, 0.4% saline, 0.3% glycine, hyaluronie acid and the like.
In a particular embodiment, the phaunaceutical compositions of the invention
comprise at least one miR gene product or miR gene expression-inhibition
compound
(or at least one nucleic acid comprising sequences encoding them) that is
resistant to
degradation by nucleases. One skilled in the art can readily synthesize
nucleic acids
that are nuclease resistant, for example, by incorporating one or more
ribonucleotides
that is modified at the 2'-position into the miR gene product. Suitable 2'-
modified
ribonucleotides include those modified at the 2'-position with fluor , amino,
alkyl,
alkoxy, and 0-allyl.
Pharmaceutical compositions of the invention can also comprise conventional
pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients
include
74

CA 02811486 2013-03-26
=
stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents.
Suitable additives include, e.g., physiologically biocompatible buffers (e.g.,
tromethamine hydrochloride), additions of chelants (such as, for example, DTPA
or
DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium
DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for
example, calcium chloride, calcium ascorbate, calcium gluconate or calcium
lactate).
Pharmaceutical compositions of the invention can be packaged for use in liquid
form,
or can be lyophilized.
For solid pharmaceutical compositions of the invention, conventional nontoxic
solid pharmaceutically-acceptable carriers can be used; for example,
pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum,
cellulose, glucose, sucrose, magnesium carbonate, and the like.
For example, a solid pharmaceutical composition for oral administration can
comprise any of the carriers and excipients listed above and 10-95%,
preferably 25%-
75%, of the at least one miR gene product or miR gene expression-inhibition
compound
(or at least one nucleic acid comprising sequences encoding them). A
pharmaceutical
composition for aerosol (inhalational) administration can comprise 0.01-20% by
weight, preferably I%-10% by weight, of the at least one miR gene product or
miR'
gene expression-inhibition compound (or at least one nucleic acid comprising
sequences encoding them) encapsulated in a liposome as described above, and a
propellant. A carrier can also be included as desired; e.g., lecithin for
intranasal
delivery.
The pharmaceutical compositions of the invention can further comprise one or
more anti-cancer agents. In a particular embodiment, the compositions comprise
at
least one miR gene product or miR gene expression-inhibition compound (or at
least
one nucleic acid comprising sequences encoding them) and at least one
chemotherapeutic agent. Chemotherapeutic agents that are suitable for the
methods of
the invention include, but are not limited to, DNA-alkylating agents, anti-
tumor
antibiotic agents, anti-metabolic agents, tubulin stabilizing agents, tubulin
destabilizing
agents, hormone antagonist agents, topoisomerase inhibitors, protein kinase
inhibitors,
HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors,
metalloproteinase inhibitors, antisense nucleic acids, triple-helix DNAs,
nucleic acid

CA 02811486 2013-03-26
aptarners, and molecularly-modified viral, bacterial and exotoxic agents.
Examples of
suitable agents for the compositions of the present invention include, but are
not limited
to, cytidine arabinoside, methotrexate, vincristine, etoposide (VP46),
doxorubicin
(adriamycin), cisplatin (CDDP), dexarnethasone, arglabin, cycIophosphamide,
sarcolysin, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU),
vinblastine,
camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin,
irinotecan,
topotecan, leucovorin, camiustine, streptozocin, CPT-11, taxol, tamoxifen,
dacarbazine,
rituxilnab, daunorubicin, 1-13-D-arabinofuranosylcytosine, imatinib,
fludarabine,
docetaxel, FOLFOX4.
The invention also encompasses methods of identifying an inhibitor of
tumorigenesis, comprising providing a test agent to a cell and measuring the
level of at
least one miR gene product in the cell. In one embodiment, the method
comprises
providing a test agent to a cell and measuring the level of at least one miR
gene product
associated with decreased expression levels in cancer cells. An increase in
the level of
the rniR gene product in the cell after the agent is provided, relative to a
suitable control'
cell (e.g., agent is not provided), is indicative of the test agent being an
inhibitor of
tumorigenesis. In a particular embodiment, at least one miR gene product
associated
with decreased expression levels in cancer cells is selected from the group
consisting of
miR-145, miR-155, miR-218-2 and combinations thereof.
In other embodiments the method comprises providing a test agent to a cell and
measuring the level of at least one miR gene product associated with increased
expression levels in cancer cells. A decrease in the level of the miR gene
product in the
cell after the agent is provided, relative to a suitable control cell(e.g.,
agent is not
provided), is indicative of the test agent being an inhibitor of
tumorigenesis. In a
particular embodiment, at least one miR gene product associated with increased
expression levels in cancer cells is selected from the group consisting of miR-
21, miR-
17-5p, miR-191, miR-29b-2, miR-223, miR-128b, miR-199a-1, miR-24-1, miR-24-2,
miR-146, miR-155, miR-181b-1, miR-20a, miR-107, miR-92-2, miR-214,
miR-30c, miR-25, miR-221, miR-106a.
Suitable agents include, but are not limited to drugs (e.g., small molecules,
peptides), and biological macromolecules (e.g., proteins, nucleic acids). The
agent can
be produced recombinantly, synthetically, or it may be isolated (i.e.,
purified) from a
76

CA 02811486 2013-03-26
=
natural source. Various methods for providing such agents to a cell (e.g.,
transfection)
are well known in the art, and several of such methods are described
hereinabove. ,
Methods for detecting the expression of at least one miR gene product (e.g.,
Northern
blotting, in situ hybridization, RT-PCR, expression profiling) are also well
known in
the art. Several of these methods are also described hereinabove.
The invention will now be illustrated by the following non-limiting examples.
EXEMPLIFICATION
The following Materials and Methods were used in the Examples:
Samples
A total of 540 samples, including 363 primary tumor samples and 177 normal
tissues, were used in this study (Table 2). The following solid cancers were
represented: lung carcinoma, breast carcinoma, prostate carcinoma, stomach
carcinoma,
colon carcinoma and pancreatic endocrine tumors. All samples were obtained
with
informed consent from each patient and were confirmed histologically. Normal
samples
were paired with samples from individuals affected with lung and stomach
carcinoma,
and from normal individuals for the remaining tissues. All normal breast
samples were
obtained by pooling 5 unrelated normal tissues. Total RNA was isolated from
tissues
using TRIzolTm reagent (Invitrogen), according to manufacturer's instructions.
MicroRNA microarrays.
Microarray analysis was performed as previously described (Liu, C.-G., et al.,
Proc. NatL Acad. Sot USA 101: 11755-11760 (2004)). Briefly, 5 jig of total RNA
was
used for hybridization on MiRNA microarray chips. These chips contain gene-
specific
40-mer oligonucleotide probes, spotted by contacting technologies and
covalently
attached to a polymeric matrix. The rnicroatrays were hybridized in 6x SSPE
(0.9 M
NaC1/60 rn_M NaH2PO4.1120/8 mM EDTA, pH 7.4)/30% fortnamide at 25 C for 18 hr,
washed in 0.75x TNT (Tris-HC1/NaC1/ Tween 20) at 37 C for 40 min, and
processed
using direct detection of the biotin-labeled transcripts by streptavidin-
Alexa647
(Molecular Probes) conjugate. Processed slides were scanned using a microarray
scanner (GenePix Pro, Axon), with the laser set to 635 run, at fixed PMT
setting and a
scan resolution of 10 mm. The data were confirmed by Northern blotting as
described
(Calm, G.A., et al., Proc. Natl. Acad. ScL USA 101:11755-11760 (2004); Iorio,
MN., et
al., Cancer Res.- 65: 7065-7070 (2005)).
77

CA 02811486 2013-03-26
=
Table 2, Samples used in the study (tumors and corresponding normals).
Tumour type Cancer Samples Normal Samples
Lung carcinoma 123 123
Breast carcinoma 79 6*
Colon carcinoma 46 ii
=
Gastric carcinoma 20 11
Endocrine pancreatic tumours 39 12
Prostate cancer 56 7
All tissues (527) 363 177
* Pools of 5 unrelated normal breast tissues per sample (for a total of 30
unrelated =
individuals).
Computational analysis.
= Microarray images were analyzed using GenePix Pro (Axon). Average values
of the replicate spots of each miRNA were background-subtracted, normali7rd
and
.subjected to further analysis. Normalization was performed by using a per
chip median
normalization method, using the median array as a reference. Finally, miRNAs
Measured as present in at least the smallest of the two classes in a dataset
were selected.
Absent calls were thresholded to 4.5 prior to statistical analysis. This level
is the
average minimum intensity level detected in the experiments. MicroRNA
nomenclature
was according to the Genome Browser and the microRNA database at Sanger Center
(Griffiths-Jones, S., Nucleic Acids Res 32: D109-11(2004)); in case of
discrepancies
we followed the microRNA database. Differentially-expressed microRNAs were
= identified by using the t test procedure within significance analysis of
microarrays
(SAM) (Tusher, V. G., et al., Proc Natl Acad Sci USA 98: 5116-21(2001). SAM
calculates a score for each gene on the basis of the change in expression
relative to
the standard deviation of all measurements. Within SAM, t test was used.
78
=
. =

CA 02811486 2013-03-26
The microRNA signatures were determined by applying nearest shrunken centroids
method. This method identifies a subgroup of genes that best characterizes
each solid
cancer from its respective normal counterpart. The prediction error was
calculated by
means of 10-fold cro- ss validation, and for each cancer, we obtained the miR
signature
that resulted in the minimal prediction error. A resampling test was performed
by
random permutation analysis to compute the p-value of the shared signature.
Example 1: Identification of a microRNA expression signature in human solid
cancers
Statistics
The combined cancers/normal tissue comparison was conducted using a
reduced number of lung samples (80 cancer and 40 normal samples), in order to
balance the different tissues numerically, yielding a total of 404 samples.
For statistical
analysis, 137 zniRs, whose expression values were above 256 (threshold value)
in at
least 50% of the samples, were retained from the 228 that were measured. A T
test was
used to identify differentially-expressed microRNAs (Table 3). The p-values of
the T
test were corrected for multiple testing procedures and to control Type I
error rates.
Adjusted p¨values were obtained by performing resampling with 500,000
permutations
(Jung, S. H., et al. Biostatistics 6: 157-69 (2005)). This analysis was
performed in
order to evaluate the results by using the same method as Lu and coworkers
(Lu, 3., et
= al., Nature 435: 834-8(2005)).
As an alternative to T test, significance analysis of microarrays (SAM) was
used
to identify differentially-expressed microRNAs. This procedure allows for the
control
of false detection rate (FDR). The delta was chosen to result in an FDR less
than or
equal to 0.01. microRNA subsets which result in the best tumor classification,
Le.,
which best predict the two classes (cancer and normal), were then identified
using the.
method of the nearest shrunken centroids, as implemented in PAM (prediction
analysis
of microarray). The prediction error was calculated by means of 10-fold cross
validation. The microRNAs were selected yielding the minimum misclassification
error
after cross-validation.
Results
By T-test, 43 differentially-expressed miRs with an adjusted p-value below
0,05
were obtained (Table 3). Twenty six miRs were overexpressed and 17 were under-
expressed relative to corresponding normal tissues when the six solid cancers
are
79

CA 02811486 2013-03-26
grouped together (breast, colon, lung, pancreas, prostate, stomach). These
results
indicated that the spectrum of expressed miRNAs in solid cancers is very
different from
that of normal cells (43 out of 137 miRNAs, 31%). Using SAM, 49 miRNAs were
identified as differentially-expressed, of which 34 were up-regulated (Table
4). Using
PAM, 36 over-expressed miRNAs in cancer (indicated by positive cancer scores)
and
21 down-regulated miRs (indicated by negative cancer scores) were identified
as
differentially-expressed (Table 5). However, these analyses are not tailored
to identify
alterations in miR expression that consistently result in transformation,
because miR
expression is heavily tissue-specific (He, L., et al. Nature 435: 828-833
(2005); also see
FIG. 1 and FIG. 2).
The clustering of miRs based on expression profiles derived from 363 solid
cancer and 177 normal samples using 228 miRs is shown in FIG. 1. The tree,
which
shows a very good separation between the different tissues, was constructed
using 137
different miRNAs that were expressed in at least 50% of the samples used in
the study..

CA 02811486 2013-03-26
.
=
t
Table 3. Differentially regulated miRs in 6 solid cancer types vs. normal
tissues (T test
= stats.) *.
Cancer Normal
miR ID Test stat Raw p Adj p
Mean Moan
,
=
,
iniR-21 1147 11.538663 9.648338 7_561136 2.00E-06 2.00E-06
milk-141 4137 '1024091 7,905398 6.2311014 2.00E46 2.000-
06
milk-212 4205 13.540651 14.33617 -6.57942 2.0013-06 2.00E-
06 .
11612-125a prte. 4113 12.32585 13.522675 -6.76358
2.000-06 2.0013-06
mill-1.38-2 11133 11.739557 13.144746 -7.01204
2.00E-06 2.00E-06
milk-218-2 422) 11.279787 12.539366 -7.40557
2.00E-06 2.00E-06
miR-23h 1151 14.169748 15.949736 -8.37744. 2.00E-06 2.00E-06
milt-195 11184 10243991 9.172985 5.763262 2.00E-06 1.00E-05
0612-212 pivc 11209 12.686966 13.661763 -5.83132
4,00E-06 1,00E-05
miR-2913-2 #95 11_27556 9.940731 5.660854
2.00E-06 1.4012-OS
iiii12-199a-1 11191 10_032002 3.920123 5_528249
2.00E-06 3.0013-05
0 tiR-9-3 1128 11.461922 12.570412 -5.43006
2.000-06 4.600-05
niiR-12.3u 11114 13.024235 13.856624 -5.35102
6.001E-06 7.20E-05
k1-73- I ilk 12.616569 13.455246 -5.35346
2.00E-06 7.20E-05
le.t-7 b 115 13-42636 14.063521 -5.17701
1.00E-05 0.000116
=
milt-16-2 1139 113.460707 9.305895 5.048375 4.00E-06
0.000224
iiiiR-199a-2 11192 9.714225 8.759237 4.862553 1.0013-05
0.000494
ill IRA 52 pAie 11151 11.388676 12.357529 -4.83716
2.00E-06 0.00053
niiR- 16-1 1138 = 10_443169 9338152 4.755258
1.00E-05 0.00071
milt-300 1172 13.982017 14.775206 -4.5707
1.20E-05 0.001476
milt-34a 1178 10.675566 9.63769 4.167301 2.60E-
05 0.00217
mi R- I 7- 5p #41 11.567244 10.281465 4.34)834
3.800-05 0_0034
milk-128h 11115 10.930395 9.047746 4.304764
3.8013-03 0.003912
miR-20a 1146 11.409852 10.19284 4304678 3.2013-05 0.003912
irti R- I Slb-1 pal: 112 I 1 9.577504 5.804294 4.225968
4.80E-05 0.004126
milt-132 11121 9.599947 8.775966 4.224737 5.60E-05
0,004(26
iiiift-200b 11195 9.475221 3.527243 .
4.221511 4_00E-05 = 0.0052 ,
let-7ii-3 114 10_436089 . 9.511546 4.03952 .
0.000104 0_003242
0612-138-1 11132 8.299613 . 9.200253 -4.05204
5_60E-05 0.00931
milt-29e 1165 11_291005 10.326912 4.019385
0_000144 0.010312
iiii R-29a 1162 11.381359 10.461075 4.013697
0.00015 0.010395
milt-96 /126 11_37212 12.136636 -3.94225
0_000138 0.012962
miR-191 11177 13.492207 12.729872 3:817222 0.0()0158 0.02015
iiiift-27a 459 10.399338 9.548582 3.715048 0.000344 0.028096
164-7g
1115 10_819688 10.01157 3.653239 0.000426 0.033874
mirt.-9- t 424 10.102819 9.212968 3-651886
0.00.0385 0.033g74
tui1k-125a 1/107 10.960998 10.005312 1651356 0.000452 0.033874
mil-95 484 9.435733 8.751331 3.59406 0.000478 0.039594
miR-155 .4157 12.505359 13.231221 -3.52369
0.000614 0.040394
iniR-199b 11194 3.755066 9.032751 3,55934 0.000588
0.04314
mi R-24-2 1154 11611696 11.612557 = 3.512774
0.00087 0.045278
(et-I111 411 12.497795 13.055093 -3.51589
0.00054 0.048354
00R-92-1 1121 16.081074 16.592426 -3.50416
0.000928 0.049828
,
* - Forty-three miRs have an adjusted p-value lower than 0.05. Twenty-six miRs
are
overexpressed and 17 down-regulated in breast, colon, lung, pancreas,
prostate,
stomach
carcinomas_
81
=

CA 02811486 2013-03-26
Table 4. Differentially regulated miRs in 6 solid cancer types vs. normal
tissues (SAM,
significance analysis of microarrays) *. .
miit ID d.value stricv p,Voltie
q.valtie R.lb Id
m 02-21 1147 3.156 0.24 0 0 2.593
' miR-2.3b 1151 -3.117 0.212 0 0 = 0.443
ni ilt- 138-2 11133 -2.514 0.2 0 0 0.402
ni IR-218-2 im 1 -1383 0.17 0 0 0384
ni ift-29h-2. 1105 2.246 0236 0 0 1.865
milt-123a pria: 11113 -2.235 0.177 0 0 0.368
milt-195 11184 2.085 0.203 0 0 1.695
111R-141 I/137 2.08 0.179 0 0 2..459
111R-199u-1 4191 1.987 0.201 0 0 1.945
in ift-9-3 #23 -1.97 0.204 0 0 0.433
milt-16-2 439 1.966 0.229 0 0 1.788
m IR- I 7-5p 441 1.964 0.296 0 0 0.725
m ift-20a 1146 1.898 0.283 0 0 0.969
milt-16-1 11311 1.57 0.232 0 0 1.417
m ilt-212 me 11209 -1.554 0.167 0 0 0.509
milt-31n 1175 1.756 0.232 0 0 1.219
tu irt-152 pree. 11151 -1.734 0.2 0 0 0.46
milt-199n-2 11192 1.721 0.196 . 0 0 1.835
n6R-12813 11115 L674 0.225 0 0 1.266
niilt-212 11203 -1.659 0.121 0 0 1627
let-711-1 #1 -1.628 0.157 0 0 1461
milt-200b 11195 1.626 0.225 0 0 1.432
mitt-1280 #114 -1.619 0.156 0 0 0.511
mik-29e 465 1.611 0.24 0 0 1.225
Int-7a-3 44 1.581 0.226 0 0 1.109
n I iR-29a 1162 1.565 0.229 0 0 1.706
milt-24-2 /154 1.555 0.284 0 0 0.831
m ift- 13S-1 11132 -1.551 0.222 0 0 0.419
milt-125u 11107 1.541 0.262 0 0
1.164 .. =
ni ilZ-106n 1199 1.511 0.275 0 0 0.952
nlift-132 11191 1.496 0.192 0 0 2.155
milt-30d /172 -1.191 0.174 0 0 0.424
milt-9-1 1124 1.478 0.244 0 0
0.763 .. .
ni i It-27u 1159 1.448 0.229 0 0 1.174
in i12.-181b- 1 pal; 11211 1_435 = 0.18 = 0 0 1.525
11 IS 1.394 0.221 0 0 1.072
milt:96 1186 -1.334 0.194 0 0 0.519
milt-191 11177 1.372 0.201 0 0 1,165
milt-93-1 1183 1.363 0.266 0 0 0.775
1161(.136 = 11130 -1.355 0.267 0 0 0.364
miR-205 11201 1.343 0.309 0 0 1.281
ni ilt-135 11(70 1.287 0.222 0.001 0.001
0.609
mill-125b- 1 11(00 1.262 1283 0.001 0.001
1.215
mi1t-10a 1130 1.252 0.227 0.001 0.001
1.643
milt-95 .1151 1_947 0.19 0.001 0-001
1.509
niiR-1991) 11194 1.22$ 0.189 0.001 0.001
1.246
miR-10b 1132 1.219 0.232 0.002 0.001
'1.312
li:t-71 1110 1.216 0.203 0.002 0.001 1.026
-5 miR-210 11205 1.213 0.237 0.002 0.001 1.058
* - Thirty five miRs are over-expressed and 14 are down-regulated in breast,
colon,
lung, pancreas, prostate, stomach carcinomas (Delta = 0.9, FDR=0.001).
. .
. 82
.. .
=

CA 02 8 114 8 6 2 0 13-0 3-2 6
'
-
Table 5. MicroRNAs selected by PAM (prediction analysis of microarray) in 6
solid
cancer types vs. normal tissues
milt 1D Solid cancer score Normal tissues score
miR-21 1147 0.0801 -0.2643
miR-138-2 4133 -0.055 0.1815
milt-218-2 11221 -0.0535 0.1765
in iR-23b 451 -0.0516 0J7
1/11R...128:i pm: 11113 -0.0498 0.1642
mill-7910-7 1195 0.0457 -0.1508
miR-195 4134 = 0.0404 -0.1333
milt-17-51) 441 0.0383 -0.1263 .
milt-9-3 = 08 -0.0357 0.1176
milt-212 pree 11209 -0.0342 0.1129
milt-213:1 446 0.0317 -0.1061
iniR-141 11137 0.0322 -0.1061
in 112-1991-1 14191 0.031.9 -0,1053
miR-I6-2 439 0.0315 -0.1037
milt-152 pr ee 4151 -0.0283 0.0933
miR-16-1 #33 0.0277 -0.0913
ntilt-34n 1178 00269 -0.0856
mill-212 4208 -0.0265 0.0375
let-73-1 41 -0.0264 0.0372
miR-12Sa 4114 -0.0259 0.0555
ittiR-12Sh 11115 0.0254 -0.0839
milt-24-2 454 0.0244 -0.0803
ntiR-29e 1165 0.0224 -0.0738 .
milt-199a-2 4192 0.0223 -0:0736
le1-71t-3 44 0.0221 -0.073
mill-191 4177 0.0185 -0.062
ittiR- 1251 4107 0.0186 410613
mill-30d 1172 -0.0185 = 0.061
milt-29a 1167 0,0184 -0.0608
milt-I 06o 1199 0_0177 -0.0584
nt112-93-1 1183 0.0163 -0.0537
milt-200h 11195 0.0159 -0.0524
10-7c.1 415 0.0158 -0.0521
mi1t-27ii 459 0.0157 -0.0518
nti12-96 486 -0.0156 0.0514
=
le1-71? 115 -0.0152 0.0501
miR-133-1 4132 -0.0151 0.0499 . =
milt-9-I 1124 0.0136 -0.0448
ntift-18 lb-1 prte 4211 0.0134 -0.0442.
naiR-155 11157 -0.0128 0.0423
.
. milt-132 4121 0.0127 -0.0418
milt-136 11130 -0.0112 0.037
let-7 i 410 0.0103 -0.034
milt-210 11205 0.0074 -0.0245
ntiR-205 4201 0.0073 -0.024
*. miR-185 4170 0.0071 -0.0234
. .
83
. .
= =-

CA 02 81148 6 2 013-03-2 6
mi R-24-1 45/ 0.007 -0.023
in 112-199h 4194 0.0054 -0.021
iniR.-125b -1 .4109 0.006 -0.0199
milt-206 pr. 4203 -0.005 0.0166
ini12-10a 430 0.0045 -0.015
484 0_0045 -0.0149
e1-7i.t 411 -0,0039 0-013
ini12-124a-3 4106 -0.0028 0.0091
iniR-101) 0.002 :0.0066
milt-185 prco. 4171 -0.0014 0.0047
miR-92-1 481 -2.00E-04 5.00E-04
* - T=1.5 and misclassification error = 0.176. Thirty six over-expressed miRs
in cancer
are indicated by positive cancer scores; 21 down-regulated miRs are indicated
by
negative cancer scores.
.5
Example 2: Identification of microRNA expression signatures associated with
various
human solid cancers.
Results
To identify microRNAs that are prognostic for cancer status associated with
solid tumors, without incurring bias due to tissue specificity, an alternative
approach
was used. First, six tissue-specific signatures, one for each cancer
histotype, were
obtained by performing independent PAM tests (summarized in Tables 6 and 7)
Specific signatures for each cancer are shown in Tables 8-13: e.g., breast-
Table 8;
colon-Table 9; lung-Table 10; pancreas-Table 11; prostate-Table 12; stomach-
Table 13.
. 15 Using these data, deregulated microRNAs that were shared among the
different
= histotype miRNA signatures were identified (Table 14). In order to
compute the p-
values for this comparative analysis, a re-sampling test with 1,000,000 random
permutations on the miRNA identity was performed. The p-value was defined as
the
relative frequency of simulation scores exceeding the real score. Twenty-one
misregulated microRNAs that were common to at least 3 types of solid cancers
(p-
value = 2.5x10-3) were identified (Table 14).
84

CA 02811486 2013-03-26
Table 6. MicroRNAs used to classify human cancers and normal tissues*.
Up- Down- Misclassification error after 10 fold
regulated regulated cross validation
Cancer miRs miRs
=
Breast 15 12 0.08
Colon 21 1 0.09
Lung 35 3 0.31
Pancreas 55 2 0.02
=
Prostate 39 6 0.11
Stomach 22 6 0.19
- Median normalization was performed and the method of the nearest shrunken
centroids was used to select predictive miRNAs.

CA 02811486 2013-03-26
=
=
Table 7. Deregulated raicroRNAs in solid common cancers*.
PAM Up- PAM Down- SAM Down-
Cancer regulated SAM Up-regulated regulated regulated
=
Breast 15 3 (FDR=0.33) 12 47
Colon 21 42 (FDR<=0.06) 1 5
Lung 35 38 (FDR<=0.01) 3 3
=
Pancreas 55 50 (FDR<=0.01) 2 8 .
Stomach 22 22 (FDR=0.06) 6 4
Prostate 39 49 (FDR=0.06) 6 3
* - Prediction analysis of microarrays (PAM) identifies those genes which best
character4e cancers and normal tissues, whilst significance analysis of
microarrays
(SAM) identifies all those whiCh have differential expression in the two
classes. False
detection rates (FDR) computed in SAM are indicated in parenthesis.
=
=
86

CA 02811486 2013-03-26
Table 8. MicroRNAs selected by prediction analysis of microarray (PAM) in
breast
. cancer (cancer vs. normal tissues) *.
miR Cancer score Normal score
mIR-21 (#47) 0.0331 -0.4364
mIR-29b-2 (#95) 0.0263 -0.3467
crydR-146 (#144) 0.0182 -0.2391
miR-125b-2 (#111) -0.0174 0.2286
= miR-125b-1 (#109) -0.0169
0.222
miR-10b (#32) -0.0164 0.2166
miR-145 (#143) -0.0158 0.2076
miR-181a (#158) 0.0153 -0.201
miR-140 (#136) -0.0122 0.1613
miR-213 (#160) 0.0116 -0.1527
=
miR-29a prec (#63) 0.0109 . -
0.1441
miR-181b-1 (#210) 0.0098 -0.1284
mIR-199b (#194) 0.0089 -0.1172
mIR-29b-1 (#64) 0.0084 -0.1111
mIR-130a (#120) -0.0076 0.1001
mIR-155 (#157) 0.0072 -0.0951
let-7a-2 (#3) -0.0042 0,0554
miR-205 (#201) -0.004 0.0533
miR-29c (#65) 0.0032 -0.0423
miR-224 (#228) -0.003 0.0399
miR-100 (#91) -0.0021 0.0283
miR-31 (#73) 0.0017 -0.022
miR-30c (#70) -7.00E-04 0.009
miR-17-5p (#41) 7.00E-04 -0.0089
miR-210 (4205) 4.00E-04 -0.0057
miR-122a (#101) 4.00E-04 -0.005
miR-16-2 (#39) -1.00E-04 0.0013
*27 miRs selected, misclassification error after cross validation of 0.008.
Seventeen
overexpressed miRs in cancer are indicated by positive cancer scores; 12 down-
regulated miRs are indicated by negative cancer scores.
=
=
87

CA 02811486 2013-03-26
Table 9. MicroRNAs selected by prediction analysis of microarray (PAM) in
colon
(cancer vs. normal tissues) *.
miR Cancer score Normal
score
miR-24-1 (#52) 0.0972 -0.5589
miR-29b-2 (#95) 0.0669 -0.3845
miR-20a (446) 0.0596 -0.3424
miR-10a (430) 0.0511 -0.2938
miR-32 (#75) 0.0401 = -0.2306
miR-203 (#197) 0.0391 -0.2251
miR-106a (#99) . 0.0364 -0.2094
miR-17-5p (#41) 0.0349 -0.2005
miR-30c (#70) 0.0328 -0.1888
miR-223 (#227) 0.0302 -0.1736
miR-126* (#102) 0Ø199 -0.1144
miR-128b (#115) 0.0177 -0.102
miR-21 (#47) 0.0162 -0.0929
=
miR-24-2 (#54) 0.0145 -0.0835
miR-99b prec (#88) 0.0125 -0.0721
miR-155 (#157) 0.0092 -0.0528
miR-213 (#160) 0.0091 -0.0522'
miR-150 (#148) 0.0042 -0.0243
miR-107 (#100) 0.003 -0.0173
miR-191 (#177) 0.0028 -0.0159
=
miR-221 (#224) 0.002 -0.0116
miR-9-3 (#28) -0.0014 0.0083
* 22 miRs selected, misclassification error after cross validation of 0.09.
Twenty-one
over-expressed miRs in cancer are indicated by positive cancer scores; I down-
regulated miR is indicated by a negative cancer score.
88
=

CA 02811486 2013-03-26
=
Table 10. MicroRNAs selected by prediction analysis of microarray (PAM) in
lung
cancer (cancer vs. normal tissues) *.
miR Cancer score Normal score
miR-21 (#47) 0.175 -0.175
miR-206 (#201) 0.1317 -0.1317
miR-200b (#195) 0.1127 -0.1127
miR-9-1 (#24) 0.1014 -0.1014 '
m1R-210 (#205) 0,0994 -0.0994
m1R-148 (#146) 0.0737 -0.0737
miR-141 (#137) 0.0631 -0.0631
miR-132 (#121) 0.0586 -0.0586
miR-215 (#213) 0.0575 -0.0575
miR-128b (#115) 0.0559 -0.0559
let-7g (#15) 0.0557 -0.0557
=
m1R-16-2 (#39) 0.0547 -0.0547
miR-129-1/2 preo (#118) 0.0515 -0.0515
miR-126* (#102) -0.0406 0.0406
miR-142-as (4139) 0.0366 -0.0366
miR-30d (#72) -0.0313 0.0313
miR-30a-5p (#66) ' -0.0297 0.0297
miR-7-2 (#21) 0.0273 4.0273
miR-199a-1 (#191) 0.0256 -0.0256
miR-127 (#112) 0.0254 -0.0254
miR-34a prec (#79) 0.0214 -0.0214
m1R-34a (#78) 0.0188 -0.0188
miR-136 (#130) 0.0174 -0.0174
m1R-202 (#196) 0.0165 -0.0165
miR-196-2 (#188) 0.0134 -0.0134
m1R-199a-2 (#192) 0.0126 -0.0126
let-72-2 (#3) 0.0109 -0.0109
m1R-124a-1 (#104) 0.0081 -0.0081
m1R-149 (#147) 0.0079 -0.0079
m1R-17-5p (#41) 0.0061 -0.0061
miR-196-1 prec (#186) 0.0053 -0.0053
miR-10a (#30) 0.0049 -0.0049
miR-99b prec (#88) 0.0045 = -0.0045
miR-196-1 (#185) 0.0044 -0.0044
miR-199b (#194) 0.0039 -0.0039 =
=
miR-191 (#177) 0.0032 -0.0032
miR-195 (#184) 7.00E-04 -7.00E-04
miti-155 (#157) 7.00E-04 -7.00E-04
* 38 miRs selected, misclassification error after cross validation of 0.31.
Thirty-five
over-expressed miRs in cancer are indicated by positive cancer scores; 3 down-
regulated miRs are indicated by negative cancer scores.
89
=

CA 02811486 2013-03-26
Table 11. MicroRNAs selected by prediction analysis of microarra:y (PAM) in
pancreatic cancer (cancer vs. normal tissues) *.
miR Cancer score Norma score
miR-103-2 (#96) 0.4746 -1.582
miR-103-1 (#97) 0.4089 -1.3631
miR-24-2 (#54) 0.4059 -1.3529
miR-107 (#100) 0.3701 -1.2336
miR-100 (#91) 0.3546 -1.182
miR-125b-2 (#111) 0.3147 -1.0489
miR-125b-1 (#109) 0.3071 -1.0237
miR-24-1 (#52) 0.2846 -0.9488
miR-191 (#177) 0.2661 -0.887
miR-23a (#50) 0.2586 -0.8619
miR-26a-1 (#56) 0.2081 -0.6937
miR-125a (#107) 0.1932 -0.644
miR-130a (#120) 0.1891 -0.6303
miR-26b (#58) 0.1861 -0.6203
miR-145 (#143) 0.1847 -0.6158
miR-221 (#224) 0.177 -0.59
miR-126* (#102) 0.1732 -0.5772
miR-16-2 (#39) 0.1698 -0.5659
miR-146 (#144) 0.1656 -0.552
miR-214 (#212) 0.1642 -0.5472
miR-99b (#89) 0.1636 -0.5454
=
miR-128b (#115) 0.1536 -0.512
miR-155 (#157) -0.1529 0.5098
miR-29b-2 (#95) - 0.1487 -0.4956
miR-29a (#62) 0.1454 -0.4848
=
=
=

CA 02811486 2013-03-26
=
Table 11 (continued). MicroRNAs selected by prediction analysis of micro array
(PAM)
in pancreatic cancer (cancer vs. normal tissues) *.
miR Cancer score Normal score
miR-25 (#55) 0.1432 -0.4775
miR-16-1 (#38) 0.1424 -0.4746
miR-99a (#90) 0.1374 -0.4581
miR-224 (#228) 0.1365 -0.4549
miR-30d (#72) 0.1301 -0.4336
miR-92-2 (#82) 0.116 -0.3865
miR-199a-1 (#191) 0.1158 -0.3861
miR-223 (#227) 0.1141 -0.3803
miR-29c (#65) 0,113 -0.3768
miR-30b (#68) 0.1008 -0.3361
miR-129-1/2 (#117) 0.1001 -0.3337
miR-197 (#189) 0.0975 -0.325
miR-17-5p (#41) 0.0955 -0.3185
miR-30c (#70) 0.0948 -0.316
miR-7-1 (#19) 0.0933 -0.311
miR-93-1 (#83) 0.0918 -0.3061
miR-140 (#136) 0.0904 -0.3015
miR-39a-5p (#66) 0.077 -0.2568
miR-132 (#121) 0.0654 -0.2179
miR-181 b-1 (#210) 0.0576 -0.1918
miR-152 prec (#151) -0.0477 0.1591
miR-23b (#51) 0,0469 -0.1562
miR-20a (#46) 0.0452 -0.1507
miR-222 (#225) 0.0416 -0.1385
miR-27a (#59) 0.0405 -0.1351
miR-92-1 (#81) 0.0332 -0.1106
rniR-21 (#47) 0.0288 -0.0959
miR-129-1/2 prec
(#118) 0.0282 -0.0939
miR-150 (#148) 0.0173 -0.0578
miR-32 (#75) 0.0167 -0.0558
miR-106a (#99) 0.0142 -0.0473
mi1-29b-1 (#64) 0.0084 -0.028
* 57 miRs selected, misclassification error after cross validation of 0.02.
Fifty-seven
miRs are over-expressed and 2 are down-regulated in cancer (indicated by
positive and
negative scores, respectively).
=
91

CA 02811486 2013-03-26
Table 12. MicroRNAs selected by prediction analysis of microarray (PAM) in
prostate
cancer (cancer vs. normal tissues) 4`.
miR Cancer score Normal
score
let-7d (#8) 0.0528 -0.4227
miR-128a prec (#113) -0.0412 0.3298
miR-195 (#184) 0.04 -0.3199
miR-203 (#197) 0.0356 " -0.2851
let-7a-2 prec (#2) -0.0313 0.2504
miR-342 (#78) 0.0303 -0.2428
miR-20a (#46) 0.029 -0.2319
miR-218-2 (#221) -0.0252 0.2018
miR-29a (#62) 0.0247 -0.1978
miR-25 (#55) 0.0233 -0.1861
miR-95 (#84) 0.0233 -0.1861
miR-197 (#189) 0.0198 -0.1587
miR-135-2 (#128) 0.0198 -0.1582
miR-187 (#173) 0.0192 -0.1535
miR-196-1 (#185) 0.0176 -0.1411
miR-148 (#146) 0.0175 -0.1401.
miR-191 (#177) 0.017 -0.136
miR-21 (#47) 0.0169 -0.1351
let-7i (#10) 0.0163 -0.1303
miR-198 (#190) 0.0145 -0.1161.
miR-199a-2 (#192) 0.0136 -0.1088
miR-30c (#70) 0.0133 -0.1062
miR-17-5p (#41) 0.0132 -0.1053
miR-92-2 (#82) 0.012 -0.0961
miR-146 (4144) 0.0113 -0_0908
miR-181b-1 prec (#211) 0.011 -0.0878
mil-32 (#75) 0.0109 -0_0873
92

CA 02811486 2013-03-26
Table 12 (continued). MicroRNAs selected by prediction analysis of micro array
(PAM)
in prostate cancer (cancer vs. normal tissues) *.
miR Cancer score Normal
score
miR-206 (#202) o.olo4 -0.083
miR-184 prec (#169) 0.0096 , -0.0764
miR-29a prec (#63) -0.0095 0.076
miR-29b-2 (#95) 0.0092 -0.0739
miR-149 (#147) -0.0084 0.0676
m iR-181b -1 (#210) 0.0049 -0.0392
miR-1 96-1 prec (#186) 0.0042 -0.0335
miR-93-1 (#83) 0.0039 -0.0312
miR-223 (#227) 0.0038 -0.0308
miR-16-1 (#38) 0.0028 -0.0226
miR-101-1 prec (#92) 0.0015 -0.0123
miR-124a-1 (#104) 0.0015 -0.0119
miR-26a-1 (#56) 0.0015 -0.0119
miR-214 (#212) 0.0013 -0.0105
miR-27a (#59) 0.0011 -0.0091
miR-24-1 (#53) -8.00E-04 0.0067
MiR-106a (#99) 7.00E-04 -0.0057
miR-199a-1 (#191). 4.00E-04 -0.0029
* - T=1, 45 miRs selected, misclassification error after cross validation of
0.11. Thirty-
nine over-expressed miRs in cancer are indicated by positive cancer scores; 6
downregulated miRs are indicated by negative cancer scores.
93

CA 02811486 2013-03-26
Table 13. MicroRNAs selected by prediction analysis of microarray (PAM) in
stomach
cancer (cancer vs. normal tissues) *.
miR Cancer score Normal
score
=
miR-223 (#227) 0.1896 -0.1806
miR-21 (#47) 0.1872 -0.1783
miR-218-2 (#221) -0.1552 0.1478
miR-103-2 (#96) " 0.1206 -0.1148
miR-92-2 (#82) 0.1142 -0.1088
miR-25 (#55) 0.1097 -0.1045
miR-136 (#130) -0.1097 0.1045
miR-191 (#177) 0.0946 -0.0901
=
miR-221 (#224) 0.0919 -0.0876
miR-125b-2 (#111) 0.0913 -0.0869
miR-103-1 (#97) 0.0837 -0.0797
miR-214 (#212) 0.0749 -0.0713
miR-222 (#225) 0.0749 -0.0713
miR-212 prec (#209) -0.054 0,0514
miR-125b-1 (#109) 0.0528 -0.0503
miR-100 (#91) 0.0526 -0.050T'
miR-107 (#100) 0.0388 -0.0369
miR-92-1 (#81) 0.0369 -0.0351
miR-96 (#86) -0.0306 0.0291
miR-192 (#178) 0.0236 -0.0224
miR-23a (#50) 0.022 -0.021
miR-215 (#213) 0.0204 -0.0194
miR-7-2 (#21) 0.0189 -0.018
miR-138-2 (#133) -0.0185 0.0176
miR-24-1 (#52) 0.0151 -0.0144
miR-99b (#89) 0.0098 -0.0093
miR-33b (#76) -0.0049 0.0046
miR-24-2 (#54) 0.0041 -0.0039
- T=1, 28 miRs selected, misclassification error after cross validation of
0.19.
Twenty-two over-expressed miRs in cancer are indicated by positive cancer
scores; 6
down-regulated miRs are indicated by negative cancer scores.
94

CA 02811486 2013-03-26
_
Table 14. The microRNAs shared by the signatures of the 6 solid cancers*.
miR N Tumor Type
miR-21 6 Breast Colon Lung Pancreas Prostate Stomach
miR-17-5p 5 Breast Colon Lung Pancreas Prostate
miR-191 5 Colon Lung Pancreas Prostate Stomach
miR-29b-2 4 Breast Colon Pancreas Prostate
miR-223 4 Colon Pancreas Prostate Stomach =
miR-128b 3 Colon Lung Pancreas
miR-199a-1 3 Lung Pancreas PrOstate
miR-24-1 3 Colon Pancreas Stomach
miR-24-2 3 Colon Pancreas Stomach
miR-146 3 Breast Pancreas Prostate
miR-155 3 Breast Colon Lung =
miR-181b-1 3 Breast Pancreas Prostate =
miR-20a 3 Colon Pancreas Prostate
miR-107 3 Colon Pancreas Stomach
miR-32 3 Colon Pancreas Prostate
miR-92-2 3 Pancreas Prostate Stomach
miR-214 3 Pancreas Prostate Stomach
miR-30c 3 Colon Pancreas Prostate
miR-25 3 Pancreas Prostate Stomach
miR-221 3 Colon Pancreas Stomach
miR-106a 3 Colon Pancreas Prostate
* - The list includes 21 commonly up-regulated microRNAs in 3 or more (N)
types of
solid cancers (p-value = 2.5x10-').
To maximize concision, the mean absolute expression levels of the deregulated
miRs for the 6 cancer/normal pairs were computed. Using the expression level
of miRs
in the comprehensive subset, the different tissues were correctly classified,
irrespective
of the disease status (FIG. 3).
FIG. 4 shows differential expression of the common microRNAs across the
different tumor tissues, in relation to the normal tissues. The tree displays
the different
cancer types according to fold changes in the miRNA subset. Prostate, colon,
stomach
and pancreatic tissues are most similar among them, while lung and breast
tissues were
represented by a fairly different signature (FIG. 4). This tree clearly shows
which
miRNAs are associated with a particular cancer histotype.
Strikingly, miR-21, miR-191 and miR-17-5p are significantly over-expressed in
all, or in 5 out of 6, of the tumor types that were considered. miR-21 was
reported to be
over-expressed in glioblastoma and to have anti-apoptotic properties (Chan,
J.A., et al.,

CA 02811486 2013-03-26
Cancer Res. 65: 6029-6033 (2005)). Lung cancer shares a portion of its
signature with
breast cancer and a portion with the other solid tumors, including miR-
17/20/92, all
three of which are members of the rnicroRNA cluster that actively cooperates
with c-
Myc to accelerate lymphomagenesis (He, L., et al., Nature 435: 828-833
(2005)). The
identification of these microRNAs as being over-expressed is an excellent
confirmation
of our approach. A second miRNA group that is activated includes miR-210 and
miR-
213, together with miR-155, which was already reported to be amplified in
large cell lymphomas (Eis, P.S., et al., Proc. Natl. Acad. Sci. USA 102: 3627-
3632
(2005)), children with Burkitt lymphoma (Metzler, M., et al., Genes
Chromosomes
Cancer 39:167-169 (2004)) and various B cell lymphomas (Kluiver, J, et al.,
Pathol.,
e-published online, July 22, 2005). These microRNAs are the only ones up-
regulated
ihi breast and lung cancer. miR-218-2 is consistently down-regulated in colon,
stomach,
prostate and pancreas cancers, but not in lung and breast carcinomas.
Several observations strengthen these results. First, in this study, the
expression
levels of both the precursor pre-miRNA and the mature miRNA were determined
for
the majority of genes. Of note, with the exception of miR-212 and miR-128a, in
all
other instances, the abnormally-expressed region was that corresponding to the
active
gene product. Second, as shown in FIG. 3, the expression variation of the
miRNAs in
the comprehensive subset was often univocal (namely, down- or up-regulation)
across
the different types of cancers, suggesting a common mechanism in human
tumorigenesis. Third, the microarray data were validated by solution
hybridization for
12 breast samples (miR-125b, miR-145 and miR-21; forio, M.V., etal., Cancer
Res. 65:
7065-7070 (2005)) and 17 endocrine pancreatic and normal samples (miR-103, miR-
155 and miR-204; data not shown), strongly confirming the accuracy of the
inicroarray
data.
Example 3: Identification ofpredicted targets for microRNAs that are
deregulated in
- solid tumors.
Materials and Methods:
Tumor suppressor and oncogene target predictions
The most recent TargetScan predictions (April 2005) were used to identify =
putative microRNA targets. These include essentially the 311.1TR targets
reported by
Lewis et al. (Lewis, B.P., et at, Cell 120: 15-20 (2005)), with a few changes
arising
96

CA 02811486 2013-03-26
from updated gene boundary definitions from the April 2005 UCSC Genome Browser
mapping of RefSeq mRNAs to the hg17 human genome assembly. Among the putative
targets, known cancer genes (tumor suppressors and oncogenes) were specified
according to their identification in the Cancer Gene Census, or as reported by
OMIM.
Target in vitro assays =
For luciferase reporter experiments, 3' UTR segments of Rbl, TGFBR2 and
Plagl that are predicted to interact with specific cancer-associated microRNAs
were
amplified by PCR from human genonaic DNA and inserted into the pGL3 control
vector (Promega) using the Xbal site immediately downstream from the stop
codon of
luciferase. The human megakaryocytic cell line, MEG-01, was grown in 10% FBS
in
=
RPM1 medium.1640, supplemented with lx nonessential nmitio acid and 1 mmol
sodium pyruvate at 37 C in a humidified atmosphere of 5% CO,. The cells were
co-
transfected in 12-well plates by using siPORT neoFX (Ambion, Austin, TX),
according
to the manufacturer's protocol, with 0.4 jig of the firefly luciferase
reporter vector and
0.08 jig of the control vector containing Renilla luciferase, pRL-TK
(Promega). For
each well, microRNA oligonucleotides (Dharmacon Research, Lafayette, CO) and
anti-
sense or scrambled oligonucleotides (Ambion) were used at a concentration of
10 nivI.
Firefly and Renilla luciferase activities were measured consecutively at 24 h
post
transfection using dual-luciferase assays (Promega).
Western blotting for RBI
Levels of RB1 protein were quantified using a mouse monoclonal anti-RBI
antibody (Santa Cruz, CA) using standard procedures for Western blotting. The
=
normalization was performed with mouse monoclonal anti-Actin antibody (Sigma).
Results
The functional significance of microRNA deregulation in cancer needs to be
understood. In solid tumors, it appears that the most common microRNA event is
gain
of expression, while loss of expression in cancer is a more limited event, and
more =
tissue specific. We used a three-step consequential approach in the following
order:
first, "in silica" prediction of targets, then luciferase assay for first
validation of cancer
relevant targets and finally, ex vivo tumor correlation between naiRNA
expression (by
97

CA 02811486 2013-03-26
microarray) and target protein expression (by Western blotting) for a specific
miRNA:mRNA interactor pair. Relevant targets for cancer miRNAs could be either
recessive (e.g., tumor suppressors) or dominant (e.g., oncogenes) cancer
genes. To test
the hypothesis that microRNAs that are deregulated in solid tumors target
known
onco genes or tumor suppressors, the predicted targets for these miRNAs were
determined using TargetS can, a database of conserved 3' UTR microRNA targets
(Lewis, B.P., et at, Cell 120: 15-20(2005)). TargetScan contained 5,121
predictions
for 18 miRNAs that are dysregulated in solid tumors, in the total 22,402
(26.5%)
predictions. One hundred fifteen out of 263 (44%) well-known cancer genes were
predicted as targets for these 18 miRNAs (Table 15). Because a high percentage
of
cancer genes are targeted by miRs that are deregulated in solid tumors, it is
unlikely
that these predictions are due to chance (P<0.0001 at Fisher exact-test).
In silico predictions for three different cancer genes, Retinoblastoma (Rb),
TGF-beta-2 receptor (TGFBR2), and pleiomorphic adenoma gene 1 (PLAG1), were
confirmed experimentally by in vitro assays. Using a luciferase reporter
assay, three
microRNAs tested (miR-106a, miR-20a and miR-26a-1) caused a significant
reduction
of protein translation relative to the scrambled control oligoRNAs in
transfected MEG-
01 cells (FIG. 6). Retinoblastoma 3'UTR, for example, was found to interact
functionally with miR-106a. The biological significance of this miRNA:mRNA
. 20 interaction is reinforced by previous reports showing that the Rbl
gene is normally
transcribed in colon cancers, whilst various fractions of cells do not express
Rbl
protein (Ali, AA., et aL, FASEB J. 7:931-937 (1993)). This finding suggests
the
existence of a post-transcriptional mechanism for regulating Rbl that could be
explained by concomitant miR-106a over-expression in colon carcinoma (FIG. 4).
Furthermore, mir-20a is down-regulated in breast cancer (FIG. 4) and TFGBR2
protein
is expressed in the epithelium of breast cancer cells (Buck, M.B., eta'.,
Clin. Cancer
Res. /0:491-498 (2004)). Conversely, the over-expression of mir-20a in colon
cancer
may represent a novel mechanism for down-regulating TGFBR2, in addition to
mutational inactivation (Biswas, S., et al., Cancer Res. 64:687-692 (2004)).
Finally, a set of patient samples was tested to verify whether RBI protein
expression correlates with miR-106a expression (FIG. 5 and FIG. 6B). As
expected, in
gastric, prostate and lung tumor samples RB1 was down-regulated (in respect to
the
98

CA 02811486 2013-03-26
=
paired normal) and miR-106a was found to be over-expressed, while in breast
tumor
samples, where miR-106a is slightly down-regulated (FIG. 5 and FIG. 6B), Rl31
is
expressed at slightly higher levels then in the paired normal control.
These experimental proofs reinforce the hypothesis that key cancer genes are
regulated by aberrant expression of miRs in solid cancers. These data add
novel
examples to the list of microRNA with important cancer gene targets, as
previously
shown by Johnsson et al. (Johnson, S.M., et al.õ Cell 120:- 635-647 (2005))
for the let-
7:Ras interaction, O'Donnell et al. (O'Donnell, LA., et al., Nature 435:839-
843
(2005)) for the miR-17-5p:cMyc interaction, and Cinimino et al. (Cimmino, A.,
et at.,
Proc. Natl. Acad. Sci. USA 102:13944-13949 (2005)) for the mir-16:Bc12
interaction.
Notably, miR-17-5p and miR-16 are members of the miRNA solid cancer signature
=
described herein.
99

CA 02811486 2013-03-26
Table 15. Oncogenes and tumor suppressor genes predicted by TargetScanS as
targets
of microRNAs from the comprehensive cancer subset*
rniRNA gene Gene Name Gene description
v-abl Abelson murine leukemia viral oricogene
miR-26a,rniR-146 ABL2 homoiog 2 (arg, Abelson-related gene= )
miR-107 AF5q31 ALL1 fused gene from 5q31
MiR-20) iniR-125b AKT3 v-akt murine thymoma viral oncogene homolog
3
miR-26a. miR-155
miR-125b APC adenomalosis polyposis coil
RHO guanine nucleotide exchange factor (GEF) 12
miR-26a, miR-218 ARHGEF12 (LARG)
miR-107. miR-221 ARNT aryl hydrocarbon receptor nuclear
translocator
miR-192 ATF1 activating transcription factor 1
Ataxia telanglectesia mutated (includes
miR-26a ATM complementation groups A. C and 0)
miR-24 AXL AXL receptor lyrosibe kinase
miR-26a, miR-107,
miR-146, miR-155
miR-138, miR-92 SCL11A B-cell CLL/Iymphoma 11A
miR-20 BCL11B B-cell CU/lymphoma 11B (CTIP2)
miR-21 BCL2 B-cell CLUlymphoma 2
rniR-26a, iniR-26a
MiFt-20, BCL6 B-cell CLUlymphorna 6 (zinc finger protein
51)
miR-92 BCL9 B-cell CLL/Iymphoma 9
miR-26a, miR-223
nliF2-221,111R-125b CBFB core-binding factor, beta subunit
= MiR-218 CCOC6 coiled-coil domain containing 6
miR-20 CCND1 cyan Di (PRAD1: parathyroid adenomatosis 1)
miR-20 CCND2 cyclin D2
miR-26a, miR-107, miR-92 CDK6 = cyclin-dependent kinase 6
=
100

CA 02811486 2013-03-26
miR-20 COKN1A cyclin-dependent kines6 inhibitor lA (p21, CIO)
miR-221, miR-92 CDKN1C cyclin-dependent kinase inhibitor 1C (p57, Kip2)
miR-24 CDX2 caudal type home box transcription factor 2
miR-92 CEBPA CCPAT/enhancer binding protein (C/EBP), alpha
MiR-26a CLIC clathrin, heavy polypeptide (He)
miR-218 COL1A1 collagen, typal, alpha 1
miR-26a CREBBP CREB binding protein (CBP)
v-cris avian sarcoma virus CT10 oncogene
miR-20 GRK homolog
miR-20 CSF1 colony stimulating factor 1 (macrophage)
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 6
MiR-221, miR-192 DOX6 (RNA helices% 54kD)
miR-138 DEK DEK oncogene (DNA binding)
miR-20 E2F1 E2F transcription factor 1
ELK3, ETS-domain protein (SRF accessory protein
miR-20 ELK3 2)
miR-24 ELL ELL gene (11-19 lysine-rich leukemia gene)
v-erb-a avian erythroblastic leukemia viral
miR-28a, miR-138 ER1BB4 oncogene homolog-like 4
MiR-221, miR-155, miR- v-ets avian erythroblastosis virus E26 oncogene
125b ETS1 homolog 1
miR-20 = E1V1 els variant gene 1
miR-1.25b Erye eta variant gene 6 (TEL oncogene)
miR-223 FAT FAT tumor suppressor (Drosophila) homolog
MiR-223. miR-125b, MiR-
218 FGFR2 fibroblast growth factor receptor 2
miR-92 FL11 Friend leukemia virus integration 1
fins-related tyrosine kinase 1 (vascular endothelial
miR-24, miR-20 FLT1 growth factor/vascular permeability factor
receptor)
v-fos FBJ murine osleosarcoma viral oncogene
MiR-221 FOS hoM0100
=
=
101
=

CA 02811486 2013-03-26
miR-92 FOXG1B forkhead box G1B
miR-223 FOX03A forkhead box 03A
miR-125b GOLGA5 golgi autoantigen, golgin subfamily a, 5 (PTC5)
miR-138 GPHN gephyrin (GPH)
miR-107, m1R-223, miR-20,
miR-218 HLF hepatic leukemia factor
miR-26a, miR-107 HMGA1 = high mobility group AT-hook 1
miR-20 HOXA13 homeo box A13
mIR-92 HOXA9 homeo box A9
miR-125b IRF4 interferon regulatory factor 4
miR-92 JUN v-jun avian Sarcoma Virus 17 oncogene homolog
v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene
miR-155 KRAS homolog
=
miR-218 LASP1 LIM and SI-13 protein 1
miR-218 LHFP lipoma HMGIC fusion partner
miR-125b, rnIR-218 LIFR leukemia inhibitory factor reteplor
miR-223 LIV102 LIM domain only 2 (rhombotin-like 1) (RBT142)
miR-223, miR-155, miR- v-mar musculoaponeurotic fibrosarcoma (avian)
125b, miR-92 MAF oncogene homolog
miR-92 MAP2K4 milogen-activaled protein kinase kinase 4
miR-146, miR-20 tvIAP3K8 mitogen-activated protein kinase kinase kinase
8
miR-125b MAX MAX protein
mill-218 tv1CC mutated in colorectal cancers
miR-24 MEN1 multiple endocrine neoplasia I
myelOidilymphoid or mixed-lineage leukemia
(trithorax homolog, Drosophila); translocated to, 6
miR-138 MLLT6 (AF17)
102
=

CA 02811486 2013-03-26
=
miR-192 IVISN = moesin
v-myb avian myeloblastosis viral oncogene
miR-24 MYB hornolog
miR-107, miR-223, m1R-146, v-myb avian myeloblastosis viral oncogene
miR-221, MiR-155, miR-218 MYBL1 hornolog-like 1
v-mye avian myelocylomalosts viral related
miR-107, miR-20 lv1YCN ancagene, neuroblastoma derived
miR-107, miR-92 MYH9 myosin, heavy polypepticle 9, non-muscle
IVIYST histone acetyltransferase (monOcylic
miR-24 MYST4 leukemia) 4 (tvlORF)
m1R-20 NBL1 neUroblastorna, suppression of tumorigenicity 1
=
miR-125b NIN ninein (GSK3B interacting protein)
miR-26a, miR-107 NKTR natural killer-tumor recognition sequence
Notch homolog 1, translocation-associated
rniR-92 NOTCH1 (Drosophila) (TAN1)
miR-24 NTRK3 neurotrophio tyrosine kinase, receptor, type 3
miR-125b PCSK7 proprotein convertase sublilisinikexin type 7
miR-24, miR-146 PERI period homolog 1 (Drosophila)
miR-146, miR-
138, PHOX2B , paired-like homeobox 2b
phosphatidylinositol binding clathrin assembly
miR-155 PICALM protein (CALM)
atiR-24, mIR-26a PIM1 pi-1 oncogene
miR-24. miR-26a, MR-21,
miR-107, miR-20, miR-155 PLAG1 pleiornorphic adenoma gene 1
miR-218 RAf38A RA88A, member MS oncogene family
v-ral simian leukemia viral oncogene hornolog A
miR-24, miR-221 RALA (lee related)
iniR-138 RARA retinoic acid receptor, alpha
miR-20, miR-192 RBI retinoblastoma 1 (including osteosarcarna)
rniR-20, RBL1 retinoblastoma-like 1 (p107)
miR-20 R13L2 retinoblestoma-like 2 (p130)
=
103

CA 02811486 2013-03-26
v-rel avian reticuloendotheflosis viral oncogene
miR-155, miR-138 REL hornotog
miR-20, miR-138 RHOC ras hornolog gene family, member C
miR-20, miR-192 R1JNX1 runt-related transcription factor 1 (AML1)
miR-107,.mIR-223 SEPT septin 6
miR-146, rniR-20, mIR-125b SET SET translocation
miR-21, miR-20, miR-155,
miR-218 SKI v-ski avian sarcoma viral oncogene homolog
SMAD, mothers against DPP homolog 4
miR-26a. rni13-146 SMAD4 (Drosophila)
spleen focus forming virus (SFFV) proviral
=
miR-155 SPII integration oncogene spit
miR-125b 6518 synovial sarcoma translocation, chromosome 18
miR-107, miR-155 SUFU suppressor of fused homolog (Drosophila)
TAPI5 RNA polyrnerase II, TATA box binding
miR-92 TAF15 protein (TBP)-associated factor, 68kDa
transcription factor 12 (HTF4, helix-loop-helix
miR-26a, miR-221, rniR-138 TCF12 transcription factors 4)
transforming growth factor, beta receptor It (70-
miR-21, miR-20 TGFEIR2 801W)
miR-24, miR-26a, miR-92 10F1 topoisomerase (DNA) I
miR-138 TPM4 tropornyosIn 4
miR-20 TRIP11 thyroid hormone receptor interaclor 11
'miR-92 TSC1 Tuberous sclerosis 1
miR-20 TSG101 Tumor susceptibility gene 101
TUSC2 Tumor suppressor candidate 2
miR-20
miR-24 VAVI vav 1 oncogene
miR-125b VAV2 vav 2 oncogene
miR-107 WEISC1 Wolf-Hirschhorn syndrome candidate i(NIMSET)
Wolf-Hirschhorn syndrome candidate 1-like I
miR-138 WHSC1L (NSD3)
104

CA 02811486 2013-03-26
(
wingless-type IvIMTV integration site famN
rn1R-28a VVNT5A member 5A
v-yes-1 Yamaguchi sarcoma viral oncogene
m113-26a, MIR-20, miR-125b. YES1 homolog 1
miR-107, miR-221 ZNFi98 zinc finger protein 198
miR-218 ZNFN1A1 zinc finger protein,
Subfamily 1A, 1 (Ikares) =.
* - Known cancer genes (e.g., tumor suppressors, oncogenes) comprise those
identified
in the
Cancer Gene Census or reported by OMIM.
While this invention has been particularly shown and described with references
to
preferred embodiments thereof, it will be understood by those skilled in the
art that various
changes in form and details may be made therein without departing from the
scope of the
invention encompassed by the appended claims.
=
=
105
. .