U.S. patent application number 14/817774 was filed with the patent office on 2015-11-19 for use of mir-26 family as a predictive marker for hepatocellular carcinoma and responsiveness to therapy.
This patent application is currently assigned to THE OHIO STATE UNIVERSITY. The applicant listed for this patent is Fudan University, The Government of the United States of America as represented by the Secretary of the Department of, The Ohio State University, The Government of the United States of America as represented by the Secretary of the Department of. Invention is credited to Carlo M. Croce, Junfang Ji, Hui-Chuan Sun, Zhao-You Tang, Xin W. Wang.
Application Number | 20150329919 14/817774 |
Document ID | / |
Family ID | 41417117 |
Filed Date | 2015-11-19 |
United States Patent
Application |
20150329919 |
Kind Code |
A1 |
Wang; Xin W. ; et
al. |
November 19, 2015 |
USE OF MiR-26 FAMILY AS A PREDICTIVE MARKER FOR HEPATOCELLULAR
CARCINOMA AND RESPONSIVENESS TO THERAPY
Abstract
Provided is a method of selecting a patient diagnosed with HCC
as a candidate for IFN-.alpha. therapy, by detecting the level of
microRNA-26 expression in a sample obtained from the patient.
Inventors: |
Wang; Xin W.; (Rockville,
MD) ; Ji; Junfang; (Bethasda, MD) ; Croce;
Carlo M.; (Columbus, OH) ; Sun; Hui-Chuan;
(Shanghai, CN) ; Tang; Zhao-You; (Shanghai,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Government of the United States of America as represented by
the Secretary of the Department of
The Ohio State University
Fudan University |
Rockville
Columbus
Shanghai |
MD
OH |
US
US
CN |
|
|
Assignee: |
THE OHIO STATE UNIVERSITY
Columbus
OH
FUDAN UNIVERSITY
Shanghai
MD
The Government of the United States of America as represented by
the Secretary of the Department of
Rockville
|
Family ID: |
41417117 |
Appl. No.: |
14/817774 |
Filed: |
August 4, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12997419 |
Jan 18, 2011 |
9125923 |
|
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PCT/US2009/046999 |
Jun 11, 2009 |
|
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14817774 |
|
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61131800 |
Jun 11, 2008 |
|
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Current U.S.
Class: |
424/85.7 ;
435/6.11; 435/6.12; 435/6.13; 435/6.14; 506/16; 506/9 |
Current CPC
Class: |
C12Q 1/6809 20130101;
A61P 35/00 20180101; C12Q 2600/112 20130101; C12Q 2600/136
20130101; C12Q 1/6809 20130101; C12Q 2600/178 20130101; A61P 1/16
20180101; C12Q 1/6886 20130101; C12Q 2600/106 20130101; A61K 31/70
20130101; A61K 38/212 20130101; C12Q 2525/207 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 38/21 20060101 A61K038/21 |
Claims
1. A method of selecting a patient diagnosed with HCC as a
candidate for IFN-.alpha. therapy, comprising detecting the level
of miR-26 expression in a HCC tumor sample obtained from the
patient, wherein a 1.5-fold or greater decrease in the level of
miR-26 expression in the tumor sample relative to a control
indicates the patient is a candidate for IFN-.alpha. therapy,
wherein expression of miR-26 in the tumor sample is decreased at
least 2-fold, at least 2.5-fold, at least 3-fold, at least
3.5-fold, or at least 4-fold.
2. A method of identifying a therapeutic agent for the treatment of
HCC, comprising screening candidate agents in vitro to select an
agent that increases expression of miR-26 in HCC cells, thereby
identifying an agent for the treatment of HCC.
3. The method of claim 1, wherein screening comprises contacting
the candidate agents with the HCC cells.
4. The method of claim 1, wherein expression of miR-26 in the HCC
cells is increased at least 2-fold relative to untreated cells.
5. The method of claim 1, wherein expression of miR-26 in the HCC
cells is increased at least 3-fold relative to untreated cells.
6. The method of claim 1, wherein expression of miR-26 in the HCC
cells is increased at least 4-fold relative to untreated cells.
7. The method of claim 1, wherein the candidate agents are
cytokines.
8. The method of claim 1, wherein the candidate agents are small
molecules.
9. The method of claim 1, wherein the HCC cells are primary cells
obtained from a HCC patient.
10. The method of claim 1, wherein the HCC cells are immortalized
cells.
11. A method of treating a patient diagnosed with HCC, comprising:
i) detecting the level of miR-26 expression in a tumor sample
obtained from the patient; ii) comparing the level of miR-26
expression in the tumor sample to a control; and iii) selecting a
method of treatment for the patient, wherein treatment comprises
IFN-.alpha. therapy only if the patient has a 1.5-fold or greater
decrease in the level of miR-26 expression in the tumor sample
relative to the control.
12. The method of claim 11, wherein miR-26 is miR-26a-1, miR-26a-2,
miR-26b, or a combination thereof.
13. The method of claim 11, wherein treatment further comprises
liver resection.
14. The method of claim 11, wherein IFN-.alpha. therapy comprises
administration of IFN-.alpha..
15. The method of claim 11, wherein the control is a non-cancerous
tissue sample obtained from the patient.
16. The method of claim 11, wherein the control is a liver sample
from a healthy subject.
17. The method of claim 11, wherein the control is a standard
value.
18. The method of claim 11, wherein expression of miR-26 in the
tumor sample is decreased at least 2-fold, at least 2.5-fold, at
least 3-fold or at least 4-fold.
19. A method for the characterization of hepatocellular carcinoma
(HCC), wherein at least one feature of HCC is selected from one or
more of the group consisting of: presence or absence of HCC;
diagnosis of HCC; prognosis of HCC; therapy outcome prediction;
therapy outcome monitoring; suitability of HCC to treatment, such
as suitability of HCC to chemotherapy treatment and/or radiotherapy
treatment; suitability of HCC to hormone treatment; suitability of
HCC for removal by invasive surgery; suitability of HCC to combined
adjuvant therapy.
20. A kit for the detection of HCC, the kit comprising at least one
detection probe comprising one or more members of the miR-26
family.
21. The kit for the detection of HCC according to claim 20, wherein
the kit is in the form or comprises an oligonucleotide array.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 12/997,419 filed Jan. 18, 2011, now allowed, which claims
priority to PCT/US2009/046999 filed Jun. 11, 2009, which claims the
benefit of U.S. Provisional Application Ser. No. 61/131,800 filed
Jun. 11, 2008, the entire disclosures of which are expressly
incorporated herein by reference.
TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION
[0002] This disclosure describes the identification of miR-26 as a
predictive marker for HCC patient prognosis and response to
interferon (IFN)-.alpha. adjunctive therapy.
BACKGROUND
[0003] Hepatocellular carcinoma (HCC) is one of the most prevalent
human malignancies worldwide, with an increasing incidence in the
United States (Parkin et al., CA Cancer J. Clin. 55(2):74-108,
2005). HCC arises most frequently in patients with inflammatory
livers resulting either from viral hepatitis caused by infection
with hepatitis B virus (HBV) or hepatitis C virus (HCV), or from
metabolic disorders or toxic insults. Viral hepatitis contributes
to over 80% of HCC cases in the world (Thorgeirsson and Grisham,
Nat. Genet. 31(4):339-3465, 2002; Budhu and Wang, J. Leukoc. Biol.
80(6):1197-1213, 2006). One of the key features of HCC is its
gender disparity with a striking male dominance (i.e. 2-6 times
more common in males than in females) (El Serag and Rudolph,
Gastroenterology 132(7):2557-2576, 2007). Classical in vivo
carcinogenesis experiments also reveal a higher susceptibility to
HCC in male rodents (Ghebranious and Sell, Hepatology
27(2):383-391, 1998; Nakatani et al., Jpn. J. Cancer Res.
92(3):249-256, 2001; Rogers et al., Cancer Res. 67(24):11536-11546,
2007; Naugler et al., Science 317(5834):121-124, 2007). Moreover,
female HCC patients tend to have a longer survival than male
patients (Ng et al., Cancer 75(1):18-22, 1995; Dohmen et al., J.
Gastroenterol. Hepatol. 18(3):267-272, 2003; Tangkijvanich et al.,
World J. Gastroenterol. 10(11):1547-1550, 2004.). These results
indicate that tumor biology and host microenvironment may differ
significantly between males and females.
[0004] A recent study suggests that the gender disparity observed
in HCC may be due to an induction of Kupffer cell-produced
interleukin-6 (IL-6), which can be inhibited by estrogen (Naugler
et al., Science 317(5834):121-124, 2007). Consistent with this
idea, other studies have revealed that serum IL-6 is highly
elevated in several aggressive malignancies including HCC, and its
expression, which can be produced by tumor cells, is associated
with metastatic diseases and poor prognosis (Ashizawa et al.,
Gastric Cancer 8(2):124-131, 2005; Porta et al., Ann. Oncol.
19(2):353-358, 2008). These studies suggest that the
procarcinogeneic activities of IL-6 may be regulated by sex
hormones and tumors with activated IL-6 may be biologically
distinct and more aggressive.
[0005] Surgery remains the only effective treatment modality for
HCC to date with a potential to cure. However, only about 10-20%
patients with HCC are currently eligible for surgical intervention.
In addition, patients who receive curative resections often have a
high frequency of relapse. Thus, a need remains to develop
diagnostic tools that provide a sufficient resolution in assisting
patient stratification for prognosis and therapy.
SUMMARY OF THE DISCLOSURE
[0006] MicroRNAs (miRs) are small, single-stranded RNA molecules
that regulate gene expression. It is disclosed herein that
expression of microR-26 (miR-26) is decreased in HCC tumor tissue
relative to non-cancerous tissue and a low level of miR-26 is
associated with a poor clinical outcome. It is also disclosed
herein that a low expression level of miR-26 is correlated with a
favorable response to interferon (IFN)-.alpha. therapy in HCC
patients. Thus, provided herein is a method of predicting the
clinical outcome of a patient diagnosed with HCC, comprising
detecting the level of miR-26 expression in a HCC tumor sample
obtained from the patient, wherein a decrease in the level of
miR-26 expression in the tumor sample relative to a control
predicts a decrease in survival, a favorable response to
IFN-.alpha. therapy, or both.
[0007] Also provided is a method of selecting a patient diagnosed
with HCC as a candidate for IFN-.alpha. therapy, comprising
detecting the level of miR-26 expression in a HCC tumor sample
obtained from the patient, wherein decrease in the level of miR-26
expression in the tumor sample relative to a control indicates the
patient is a candidate for IFN-.alpha. therapy.
[0008] Further provided is a method of treating a patient diagnosed
with HCC, comprising (i) detecting the level of miR-26 expression
in a tumor sample obtained from the patient; (ii) comparing the
level of miR-26 expression in the tumor sample to a control; and
(iii) selecting a method of treatment for the patient, wherein
treatment comprises IFN-.alpha. therapy only if the patient has a
1.5-fold or greater decrease in the level of miR-26 expression in
the tumor sample relative to the control.
[0009] In some embodiments of the methods provided herein, the
control is a non-cancerous tissue sample obtained from the patient.
In other embodiments, the control is a liver sample from a healthy
subject or a standard value.
[0010] Further provided is a method of identifying a therapeutic
agent for the treatment of HCC, comprising screening candidate
agents in vitro to select an agent that increases expression of
miR-26 in HCC cells, thereby identifying an agent for the treatment
of HCC. In some embodiments, screening comprises contacting the
candidate agents with the HCC cells. The candidate agents can be
any type of molecule, including, but not limited to cytokines or
small molecules.
[0011] The foregoing and other features and advantages of the
disclosure will become more apparent from the following detailed
description of several embodiments which proceeds with reference to
the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIGS. 1A-1D: miR-26 expression in male and female hepatic
tissues and tumors.
[0013] FIG. 1A: The expression levels of miR-26a-1 in female (n=30)
and male non-cancerous hepatic tissues (n=194), determined by
microarray analysis. An un-paired t-test was used.
[0014] FIG. 1B: The relative expression levels of miR-26a from
female cases (n=26) and age-matched male G1 and G2 cases (n=56)
were determined by qRT-PCR. U n-paired t-tests were used.
[0015] FIG. 1C: Comparisons of relative levels of miR-26a-1 between
224 paired NT and T tissues when dichotomized by miR-26 status in
tumors. Paired t-tests were used: p<0.001 from the low miR-26-1
group; p=0.23 from the high miR-26-1 group. The data in FIGS. 1A-1C
are expressed as log 2 relative expression normalized to a
disease-free normal liver pool (n=8).
[0016] FIG. 1D: miR-26a-1 expression levels in tumors, determined
by microarray analysis, and survival outcomes. A log rank test was
used and a median expression level was used as a cutoff. Low miR-26
expression (n=106) was classified as the lower 50th percentile
(with a mean 2.69-fold reduction in T compared to NT). High miR-26
expression (n=111) was classified as the upper 50th percentile
(with a mean 0.98-fold reduction in T compared to NT).
[0017] FIGS. 2A-2C: Distinct transcriptional activities in low
miR-26 HCCs.
[0018] FIG. 2A: A multidimensional scaling plot of 224 HCC cases
based on the expression of 11,580 genes. Samples are colored based
on median dichotomization into low miR-26 expression (blue or
light) or high miR-26 expression (red or dark).
[0019] FIG. 2B: A Venn diagram of mRNAs coexpressed in low miR-26
HCCs.
[0020] FIG. 2C: Gene networks of NFkB/IL-6 signaling in low miR-26
HCCs. Upregulated genes in low miR-26 HCC are highlighted in orange
or light gray. Genes in gray print are not on the significant gene
list but are reported to be associated with the network. Solid
lines and dotted lines represent direct and indirect interactions,
respectively, while arrows represent positive regulation (i.e.,
acts on) of gene expression. Genes connected by lines represent
binding only. Detailed network relationships and network shapes are
described in FIG. 15.
[0021] FIGS. 3A-3F: The association of miR-26a expression in tumors
with survival prognosis in two prospective randomized control
trials with IFN treatment (IFN test cohort, n=118; IFN validation
cohort, n=79).
[0022] FIGS. 3A-3B: The association of miR-26a expression with
overall survival in HCC patients from control groups (cohort 2,
panel FIG. 3A; cohort 3, panel FIG. 3B). Cohort 2: high miR-26a
cases, n=24; low miR-26a cases, n=35. Cohort 3: high miR-26a cases,
n=21; low miR-26a cases, n=19.
[0023] FIGS. 3C-3D: The association of IFN adjuvant therapy with
overall survival in HCC patients with low miR-26a expression.
Cohort 2: IFN cases, n=24; control cases, n=35. Cohort 3: IFN
cases, n=20; control cases, n=19.
[0024] FIGS. 3E-3F: The association of IFN adjuvant therapy with
overall survival in HCC patients with high miR-26a expression.
Cohort 2: IFN cases, n=35; control cases, n=24. Cohort 3: IFN
cases, n=19; control cases, n=21.
[0025] FIG. 4A: Table 1--Clinical characteristics of the subjects
for Cohort 1 and Cohort 2.
[0026] FIG. 4B: Supplemental Table 1--Clinical characteristics of
the subjects for INF tests--Cohort 2 and Cohort 3.
[0027] FIG. 4C: Supplemental Table 2--Clinical characteristics of
cases used to search for gender-related microRNAs.
[0028] FIG. 5A: Table 2--Univariate and Multivariate Cox Regression
Analysis of miR-26 Expression Levels and Overall Survival in
Subjects with HCC.
[0029] FIG. 5B: Table 3--Univariate and Multivariate Cox Regression
Analysis of Interferon Therapy and Overall Cancer Survival in
Subjects with Low miR-26 Expression.
[0030] FIG. 6A: Supplemental Table 3--Eight Gender-Related
microRNAs.
[0031] FIG. 6B: Table 4--Top 20 list of gene networks from
INGENUITY Pathway Analysis.
[0032] FIGS. 7A-7F: The abundance of miR-26 expression in male and
female hepatic tissues and tumors from the test cohort.
[0033] FIG. 7A: The expression levels of miR-26a-2 in female (n=30)
and male non-tumor hepatic tissues (n=194).
[0034] FIG. 7B: Comparisons of relative levels of miR-26a-2 between
paired T and NT when dichotomized by miR-26 status. A median
expression level was used as a cutoff. Low miR-26 expression was
classified as the lower 50th percentile (with a mean 2.69-fold
reduction in T compared to NT). High miR-26 expression was
classified as the upper 50th percentile (with a mean 0.98-fold
reduction in T compared to NT). The data in FIG. 7A and FIG. 7B
were determined by microarray analysis and expressed as log 2
relative expression normalized to a disease-free normal liver pool
(n=8).
[0035] FIG. 7C: miR-26a-2 expression levels in tumors and survival
outcomes.
[0036] FIGS. 7D-7F: Similar results as in FIG. 7A-7C with miR-26b
expression status.
[0037] FIG. 8: Sample stratification based on miR-26 expression
status. HCC samples were classified based on their average microRNA
expression (i.e., miR-26a-1, 26a-2, and 26b). Median expression was
used to separate cases into low miR-26 (blue or light) and high
miR-26 (red or dark). The position of each dot (case) is determined
by three microRNA expression levels (miR-26a-1, miR-26a-2 and
miR-26b). The stratification outcomes were used to generate the MDS
plot.
[0038] FIGS. 9A-9B: Expression of S100P and SLC2A6 in HCC and their
correlation between microarray and qRT-PCR. Expression levels of
S100P (FIG. 9A) and SLC2A6 (FIG. 9B) in 10 HCCs with low-miR-26
level and 10 HCC cases with high miR-26 level determined by qRT-PCR
(left panel). A linear regression and correlation among data from
qRT-PCR versus microarray is shown with r (spearman) and p-value
indicated (right panel). Expression status is shown as the tumor
(T)/non-tumor (NT) ratio.
[0039] FIGS. 10A-10C: Expression of miR-26 and IL-6 in HCC.
[0040] FIG. 10A: Expression levels of IL-6 in 82 paired tumors (T)
and non-tumor tissues (NT) determined by qRT-PCR. Student's t-test
was performed to examine the IL-6 differential expression between T
and NT.
[0041] FIGS. 10B-10C: Correlation of expression levels between IL-6
and miR-26a (B FIG. 10B) or miR-26b (FIG. 10C) in 82 paired tumors
and non-tumor tissues determined by qRT-PCR. The data are shown as
the T/NT ratio on a log 2 scale.
[0042] FIG. 11: The abundance of miR-26 expression in male and
female hepatic tissues and tumors from a validation cohort.
Decreased expression of miR-26a and miR-26b in tumors (right
panels) with a more abundant expression in female than male
non-tumor tissues (left panels) is validated in an independent
validation cohort from a retrospective randomized clinical trial.
Expression levels of miR-26a and miR-26b were measured by qRT-PCR.
P values are from un-paired t-tests.
[0043] FIGS. 12A-12F: The association of miR-26b expression in
tumors with survival prognosis in two prospective randomized
control trials for IFN adjuvant therapy.
[0044] FIGS. 12A-12B: The association of miR-26b expression with
overall survival in control cases from cohort 2 (FIG. 6A) or cohort
3 (FIG. 12B). Cohort 2: high miR-26b cases, n=23; low miR-26b
cases, n=36. Cohort 3: high miR-26b cases, n=21; low miR-26b cases,
n=19.
[0045] FIGS. 12C-12D: The association of IFN adjuvant therapy with
overall survival in HCC patients with low miR-26b expression.
Cohort 2: IFN cases, n=22; control cases, n=36. Cohort 3: IFN
cases, n=20; control cases, n=19.
[0046] FIGS. 12E-12F: The association of IFN adjuvant therapy with
overall survival in HCC patients with high miR-26a expression.
Cohort 2: IFN cases, n=37; control cases, n=23. Cohort 3: IFN
cases, n=19; control cases, n=21.
[0047] FIG. 13: Expression of miR-26 in various cell types
including primary freshly isolated hepatocytes, hTERT-immortalized
normal hepatocyte cell line HHT4, two HCC cell lines and PBMC from
health donors determined by qRT-PCR.
[0048] FIG. 14: Expression of HGF in HCC and association with
miR-26. Expression levels of HGF in 10 HCCs with low-miR-26 level
and 10 HCC cases with high miR-26 level determined by qRT-PCR (left
panel). A linear regression and correlation between HGF level from
qRT-PCR and miR-26 levels from microarray is shown with r
(spearman) and p-value indicated (right panel). Expression status
is shown as the tumor (T)/non-tumor (NT) ratio.
[0049] FIG. 15: A detailed description of the network relationships
and network shapes used in the Pathway Analysis.
SEQUENCE LISTING
[0050] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand. In
the accompanying sequence listing:
[0051] [SEQ ID NO: 1] is the nucleotide sequence of the precursor
form of human miR-26a-1=guggccucgu ucaaguaauc caggauaggc ugugcagguc
ccaaugggcc uauucuuggu
[0052] [SEQ ID NO: 2] is the nucleotide sequence of the precursor
form of human miR-26a-2=ggcuguggcu ggauucaagu aauccaggau aggcuguuuc
caucugugag gccuauucuu gauuacuugu uucuggaggc agcu
[0053] [SEQ ID NO: 3] is the nucleotide sequence of the precursor
form of human miR-26b=ccgggaccca guucaaguaa uucaggauag guugugugcu
guccagccug uucuccauua cuuggcucgg ggaccgg
[0054] [SEQ ID NO: 4] is the nucleotide sequence of the mature form
of human miR-26a-1 and miR-26a-2=uucaaguaau ccaggauagg cu.
[0055] [SEQ ID NO: 5] is the nucleotide sequence of the mature form
of human miR-26b=uucaaguaau ucaggauagg u.
DETAILED DESCRIPTION
Abbreviations
[0056] 1NN 1-Nearest neighbor
[0057] 3NN 3-Nearest neighbor
[0058] AFP Alpha-fetoprotein
[0059] ALT Alanine aminotransferase
[0060] CCP Compound covariate predictor
[0061] DLD Diagonal linear discriminant
[0062] DNA Deoxyribonucleic acid
[0063] HBV Hepatitis B virus
[0064] HCC Hepatocellular carcinoma
[0065] HCV Hepatitis C virus
[0066] IFN Interferon
[0067] IL Interleukin
[0068] ISH In situ hybridization
[0069] miR MicroRNA
[0070] miRNA MicroRNA
[0071] mRNA Messenger RNA
[0072] NC Nearest centroid
[0073] PCR Polymerase chain reaction
[0074] pre-miRNA Precursor microRNA
[0075] qRT-PCR Quantitative reverse transcriptase polymerase chain
reaction
[0076] RNA Ribonucleic acid
[0077] siRNA Small interfering RNA
[0078] snRNA Small nuclear RNA
[0079] SVM Support vector machines
[0080] TACE Transcatheter arterial chemoembolization
[0081] TNM Tumor-node-metastasis
Terms
[0082] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not intended to limit the scope of the
current teachings. In this application, the use of the singular
includes the plural unless specifically stated otherwise.
[0083] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0084] Also, the use of "comprise", "contain", and "include", or
modifications of those root words, for example but not limited to,
"comprises", "contained", and "including", are not intended to be
limiting. The term "and/or" means that the terms before and after
can be taken together or separately. For illustration purposes, but
not as a limitation, "X and/or Y" can mean "X" or "Y" or "X and
Y".
[0085] It is understood that an miRNA is derived from genomic
sequences or a gene. In this respect, the term "gene" is used for
simplicity to refer to the genomic sequence encoding the precursor
miRNA for a given miRNA. However, embodiments of the invention may
involve genomic sequences of a miRNA that are involved in its
expression, such as a promoter or other regulatory sequences.
[0086] The term "miRNA" generally refers to a single-stranded
molecule, but in specific embodiments, molecules implemented in the
invention will also encompass a region or an additional strand that
is partially (between 10 and 50% complementary across length of
strand), substantially (greater than 50% but less than 100%
complementary across length of strand) or fully complementary to
another region of the same single-stranded molecule or to another
nucleic acid. Thus, nucleic acids may encompass a molecule that
comprises one or more complementary or self-complementary strand(s)
or "complement(s)" of a particular sequence comprising a molecule.
For example, precursor miRNA may have a self-complementary region,
which is up to 100% complementary miRNA probes of the invention can
be or be at least 60, 65, 70, 75, 80, 85, 90, 95, or 100%
complementary to their target.
[0087] The term "combinations thereof" as used herein refers to all
permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, ACB,
CBA, BCA, BAC, or CAB.
[0088] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the described
subject matter in any way. All literature and similar materials
cited in this application, including patents, patent applications,
articles, books, treatises, and internet web pages are expressly
incorporated by reference in their entirety for any purpose. In the
event that one or more of the incorporated literature and similar
materials defines or uses a term in such a way that it contradicts
that term's definition in this application, this application
controls.
[0089] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0090] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0091] Adjunctive therapy: A treatment used in combination with a
primary treatment to improve the effects of the primary treatment.
For example, a patient diagnosed with HCC may undergo liver
resection as a primary treatment and interferon (IFN)-.alpha.
therapy as an adjunctive therapy.
[0092] Candidate: As used herein, a "candidate" for IFN-.alpha.
therapy is a patient that is predicted to respond favorably to
IFN-.alpha. therapy for the treatment of HCC.
[0093] Clinical outcome: Refers to the health status of a patient
following treatment for a disease or disorder, such as HCC, or in
the absence of treatment. Clinical outcomes include, but are not
limited to, an increase in the length of time until death, a
decrease in the length of time until death, an increase in the
chance of survival, an increase in the risk of death, survival,
disease-free survival, chronic disease, metastasis, advanced or
aggressive disease, disease recurrence, death, and favorable or
poor response to therapy.
[0094] Control: A "control" refers to a sample or standard used for
comparison with an experimental sample, such as a tumor sample
obtained from a HCC patient. In some embodiments, the control is a
liver sample obtained from a healthy patient or a non-cancerous
tissue sample obtained from a patient diagnosed with HCC. In some
embodiments, the control is a historical control or standard value
(i.e. a previously tested control sample or group of samples that
represent baseline or normal values, such as the level of miR-26
expression in non-cancerous tissue).
[0095] Cytokines: Proteins produced by a wide variety of
hematopoietic and non-hematopoietic cells that affect the behavior
of other cells. Cytokines are important for both the innate and
adaptive immune responses.
[0096] Decrease in survival: As used herein, "decrease in survival"
refers to a decrease in the length of time before death of a
patient, or an increase in the risk of death for the patient.
[0097] Detecting level of expression: As used herein, "detecting
the level of miR-26 expression" refers to quantifying the amount of
miR-26 present in a sample. Detecting expression of miR-26, or any
microRNA, can be achieved using any method known in the art or
described herein, such as by qRT-PCR. Detecting expression of
miR-26 includes detecting expression of either a mature form of
miR-26 or a precursor form that is correlated with miR-26
expression. Typically, miRNA detection methods involve sequence
specific detection, such as by RT-PCR. miR-26-specific primers and
probes can be designed using the precursor and mature miR-26
nucleic acid sequences, which are known in the art and provided
herein as SEQ ID NOs: 1-5.
[0098] Hepatocellular carcinoma (HCC): HCC is a primary malignancy
of the liver typically occurring in patients with inflammatory
livers resulting from viral hepatitis, liver toxins or hepatic
cirrhosis (often caused by alcoholism).
[0099] Interferon (IFN)-.alpha.: Interferons are a type of cytokine
produced by a variety of different cell types, including leukocytes
(such as T cells, B cells and natural killer cells) and
fibroblasts. Interferon is induced in response to exposure to
foreign agents such as viruses, parasites and tumors.
Double-stranded RNA, often indicative of a viral infection, is a
common inducer of interferon. Interferons are important for
inhibiting viral replication, activating natural killer cells and
macrophages, and increasing antigen presentation to lymphocytes.
Interferons include IFN-.alpha., IFN-.beta. and IFN-.gamma.. As
used herein, "IFN therapy" or "IFN-.alpha. therapy" for HCC refers
to treatment with IFN-.alpha.. As used herein, "a favorable
response to IFN therapy" or "a favorable response to IFN-.alpha.
therapy" means a patient treated with IFN-.alpha. has an increase
in survival (an increase in the length of time until death, or an
increased chance of survival), an improvement in the symptoms of
HCC, a decrease in spread or metastasis of HCC, a decrease in
severity or aggressiveness of disease, or any other appropriate
clinical parameter for measuring a positive response to
therapy.
[0100] MicroRNA (miRNA, miR): Single-stranded RNA molecules that
regulate gene expression. MicroRNAs are generally 21-23 nucleotides
in length. MicroRNAs are processed from primary transcripts known
as pri-miRNA to short stem-loop structures called precursor
(pre)-miRNA and finally to functional, mature microRNA. Mature
microRNA molecules are partially complementary to one or more
messenger RNA molecules, and their primary function is to
down-regulate gene expression. MicroRNAs regulate gene expression
through the RNAi pathway.
[0101] MicroRNA-26: Refers to a family of microRNAs (also referred
to as miRs) that currently includes miR-26a-1, miR-26a-2 and
miR-26b. The term "microRNA-26" also includes any as yet
unidentified members of the microRNA-26 family that are
differentially expressed in HCC tumors relative to healthy
tissues.
[0102] miR-26 expression: As used herein, "low miR-26 expression"
and "high miR-26 expression" are relative terms that refer to the
level of miR-26 found in a sample, such as a healthy or HCC liver
sample. In some embodiments, low and high miR-26 expression are
determined by comparison of miR-26 levels in a group of
non-cancerous and HCC liver samples. Low and high expression can
then be assigned to each sample based on whether the expression of
miR-26 in a sample is above (high) or below (low) the average or
median miR-26 expression level. For individual samples, high or low
miR-26 expression can be determined by comparison of the sample to
a control or reference sample known to have high or low expression,
or by comparison to a standard value. Low and high miR-26
expression can include expression of either the precursor or mature
forms or miR-26, or both.
[0103] Patient: As used herein, the term "patient" includes human
and non-human animals. The preferred patient for treatment is a
human. "Patient" and "subject" are used interchangeably herein.
[0104] Pharmaceutically acceptable vehicles: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of one or more therapeutic compounds, molecules or agents.
[0105] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0106] Preventing, treating or ameliorating a disease: "Preventing"
a disease (such as HCC) refers to inhibiting the full development
of a disease. "Treating" refers to a therapeutic intervention that
ameliorates a sign or symptom of a disease or pathological
condition after it has begun to develop. "Ameliorating" refers to
the reduction in the number or severity of signs or symptoms of a
disease.
[0107] Screening: As used herein, "screening" refers to the process
used to evaluate and identify candidate agents that increase
expression of miR-26. In some cases, screening involves contacting
a candidate agent (such as an antibody, small molecule or cytokine)
with HCC cells and testing the effect of the agent on expression of
miR-26. Expression of a microRNA can be quantified using any one of
a number of techniques known in the art and described herein, such
as by microarray analysis or by qRT-PCR.
[0108] Small molecule: A molecule, typically with a molecular
weight less than about 1000 Daltons, or in some embodiments, less
than about 500 Daltons, wherein the molecule is capable of
modulating, to some measurable extent, an activity of a target
molecule.
[0109] Therapeutic: A generic term that includes both diagnosis and
treatment.
[0110] Therapeutic agent: A chemical compound, small molecule, or
other composition, such as an antisense compound, antibody,
protease inhibitor, hormone, chemokine or cytokine, capable of
inducing a desired therapeutic or prophylactic effect when properly
administered to a subject. For example, therapeutic agents for HCC
include agents that prevent or inhibit development or metastasis of
HCC. As used herein, a "candidate agent" is a compound selected for
screening to determine if it can function as a therapeutic agent
for HCC. In some embodiments, the candidate agent is identified as
a therapeutic agent if the agent increases expression of miR-26 in
HCC cells. "Incubating" includes a sufficient amount of time for an
agent to interact with a cell or tissue. "Contacting" includes
incubating an agent in solid or in liquid form with a cell or
tissue. "Treating" a cell or tissue with an agent includes
contacting or incubating the agent with the cell or tissue.
[0111] Therapeutically effective amount: A quantity of a specified
pharmaceutical or therapeutic agent sufficient to achieve a desired
effect in a subject, or in a cell, being treated with the agent.
For example, this can be the amount of a therapeutic agent that
increases expression of miR-26 and/or the amount of a therapeutic
agent that prevents, treats or ameliorates HCC in a patient. The
effective amount of the agent will be dependent on several factors,
including, but not limited to the subject or cells being treated,
and the manner of administration of the therapeutic
composition.
[0112] Tumor, neoplasia, malignancy or cancer: The result of
abnormal and uncontrolled growth of cells. Neoplasia, malignancy,
cancer and tumor are often used interchangeably and refer to
abnormal growth of a tissue or cells that results from excessive
cell division. The amount of a tumor in an individual is the "tumor
burden" which can be measured as the number, volume, or weight of
the tumor. A tumor that does not metastasize is referred to as
"benign." A tumor that invades the surrounding tissue and/or can
metastasize is referred to as "malignant."
[0113] Tumor-Node-Metastasis (TNM): The TNM classification of
malignant tumors is a cancer staging system for describing the
extent of cancer in a patient's body. T describes the size of the
primary tumor and whether it has invaded nearby tissue; N describes
any lymph nodes that are involved; and M describes metastasis. TNM
is developed and maintained by the International Union Against
Cancer to achieve consensus on one globally recognized standard for
classifying the extent of spread of cancer. The TNM classification
is also used by the American Joint Committee on Cancer and the
International Federation of Gynecology and Obstetrics.
[0114] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present disclosure, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including explanations of terms, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
Overview of Several Embodiments
[0115] It is disclosed herein that expression of miR-26 is
decreased in HCC tumor tissue relative to non-cancerous tissue, and
a low level of miR-26 is associated with a poor clinical outcome.
It is also disclosed herein that a low expression level of miR-26
is correlated with a favorable response to IFN-.alpha. therapy in
HCC patients.
[0116] Thus, provided herein is a method of predicting the clinical
outcome of a patient diagnosed with HCC, comprising detecting the
level of miR-26 expression in a HCC tumor sample obtained from the
patient, wherein a decrease in the level of miR-26 expression in
the tumor sample relative to a control predicts a decrease in
survival, a favorable response to IFN-.alpha. therapy, or both.
Also provided is a method of selecting a patient diagnosed with HCC
as a candidate for IFN-.alpha. therapy, comprising detecting the
level of miR-26 expression in a HCC tumor sample obtained from the
patient, wherein a decrease in the level of miR-26 expression in
the tumor sample relative to a control indicates the patient is a
candidate for IFN-.alpha. therapy.
[0117] In one embodiment of the methods, the miR-26 is miR-26a-1.
In another embodiment, the miR-26 is miR-26a-2. In another
embodiment, the miR-26 is miR-26b. In other embodiments, miR-26 is
a combination of two or more of miR-26a-1, miR-26a-2 and
miR-26b.
[0118] In some embodiments, the control is non-cancerous tissue
sample obtained from the same patient. In other embodiments, the
control is a liver sample obtained from a healthy subject, such as
a healthy liver donor. In another example, the control is a
standard calculated from historical values. Tumor samples and
non-cancerous tissue samples can be obtained according to any
method known in the art. For example, tumor and non-cancerous
samples can be obtained from HCC patients that have undergone liver
resection, or they can be obtained by extraction using a hypodermic
needle, by microdissection, or by laser capture. Control
(non-cancerous) samples can be obtained, for example, from a
cadaveric donor or from a healthy liver donor.
[0119] Expression of miR-26 in the tumor sample is decreased
(relative to a control) by an amount sufficient to impart a
phenotypic effect, such as rendering the tumor more susceptible to
treatment by IFN-.alpha., altering the rate of growth of the tumor,
rendering the tumor capable of metastasis. While not wishing to be
bound by theory, the phenotypic effect is thought to be mediated by
differential gene expression regulated by miR-26. The phenotypic
effect can also be an increase or decrease in expression of one or
more miR-26 regulated genes. In some embodiments, expression of
miR-26 in the tumor sample is decreased at least 1.1-fold, at least
1.2-fold, at least 1.3-fold or at least 1.4-fold. In one
embodiment, expression of miR-26 in the tumor sample is decreased
at least 1.5-fold relative to the control. In another embodiment,
expression of miR-26 in the tumor sample is decreased at least
2-fold relative to the control. In another embodiment, expression
of miR-26 in the tumor sample is decreased at least 2.5-fold
relative to the control. In another embodiment, expression of
miR-26 in the tumor sample is decreased at least 3-fold relative to
the control. In another embodiment, expression of miR-26 in the
tumor sample is decreased at least 3.5-fold relative to the
control. In another embodiment, expression of miR-26 in the tumor
sample is decreased at least 4-fold relative to the control. In
other embodiments, expression of miR-26 in the tumor sample is
decreased at least 5-fold, at least 6-fold, at least 7-fold, at
least 8-fold, at least 9-fold, or at least 10-fold. Expression of
miR-26 can be detected and quantified using any method known in the
art, such as, but not limited to microarray and qRT-PCR.
[0120] Further provided is a method of identifying a therapeutic
agent for the treatment of HCC, comprising screening candidate
agents in vitro to select an agent that increases expression of
miR-26 in HCC cells, thereby identifying an agent for the treatment
of HCC.
[0121] In some embodiments, screening comprises contacting the
candidate agents with the HCC cells. The HCC cells can be primary
cells obtained from a HCC patient, or the HCC cells can be
immortalized or transformed cells. In one embodiment, expression of
miR-26 in the HCC cells is increased at least 2-fold relative to
untreated cells. In another embodiment, expression of miR-26 in the
HCC cells is increased at least 3-fold relative to untreated cells.
In another embodiment, expression of miR-26 in the HCC cells is
increased at least 4-fold relative to untreated cells.
[0122] The candidate agents can be any type of agent, such as a
protein, peptide, small molecule, antibody or nucleic acid. In some
embodiments, the candidate agent is a cytokine. In some
embodiments, the candidate agent is a small molecule. Screening
includes both high-throughout screening and screening individual or
small groups of candidate agents.
[0123] Further provided is a method of treating a patient diagnosed
with HCC, comprising (i) detecting the level of miR-26 expression
in a tumor sample obtained from the patient; (ii) comparing the
level of miR-26 expression in the tumor sample to a control; and
(iii) selecting a method of treatment for the patient, wherein
treatment comprises IFN-.alpha. therapy only if the patient has a
1.5-fold or greater decrease in the level of miR-26 expression in
the tumor sample relative to the control. In some embodiments, the
miR-26 is miR-26a-1, miR-26a-2, miR-26b, or a combination
thereof.
[0124] In some embodiments, the method of treatment further
comprises liver resection. In some embodiments, IFN-.alpha. therapy
comprises administration of IFN-.alpha..
[0125] In one embodiment, the control is a non-cancerous tissue
sample obtained from the patient. In another embodiment, the
control is a liver sample from a healthy subject. In another
embodiment, the control is a standard value.
[0126] In some embodiments of the method, expression of miR-26 in
the tumor sample is decreased at least 2-fold, at least 2.5-fold,
at least 3-fold or at least 4-fold.
[0127] Tumor Tissue Samples
[0128] The methods provided herein include detecting the level of
miR-26 expression in tumor and non-tumor tissue samples. In some
embodiments, the tissue samples are obtained from subjects
diagnosed with HCC and, in some cases, from healthy subjects or
cadaveric donors. A "sample" refers to part of a tissue that is
either the entire tissue, or a diseased or healthy portion of the
tissue. As described herein, tumor tissue samples are compared to a
control. In some embodiments, the control is non-cancerous tissue
sample obtained from the same subject, such as non-cancerous
hepatic tissue surrounding the HCC tumor. In other embodiments, the
control is a liver sample obtained from a healthy patient or a
non-cancerous tissue sample from a cadaver. In other embodiments,
the reference sample is a standard based on historical values.
[0129] Tissue samples can be obtained from a subject using any
method known in the art. For example, tissue samples can be
obtained from HCC patients who have undergone liver resection as a
treatment for HCC. From these patients, both tumor tissue and
surrounding non-cancerous hepatic tissue can be obtained. In some
embodiments, the non-cancerous tissue sample used as a control is
obtained from a cadaver. In other embodiments, the non-cancerous
tissue sample is obtained from a healthy liver donor (see Kim et
al., Hepatology 39(2):518-527, 2004).
[0130] In some embodiments, tissue samples are obtained by biopsy.
Biopsy samples can be fresh, frozen or fixed, such as
formalin-fixed and paraffin embedded. Samples can be removed from a
patient surgically, by extraction (for example by hypodermic or
other types of needles), by microdissection, by laser capture, or
by any other means known in the art.
[0131] Methods of Detecting miR-26 Expression
[0132] The sequences of precursor microRNAs (pre-miRNAs) and mature
miRNAs are publicly available, such as through the miRBase
database, available online by the Sanger Institute (see
Griffiths-Jones et al., Nucleic Acids Res. 36:D154-D158, 2008;
Griffiths-Jones et al., Nucleic Acids Res. 34:D140-D144, 2006; and
Griffiths-Jones, Nucleic Acids Res. 32:D109-D111, 2004). The
sequences of the precursor and mature forms of miR-26 family
members are provided herein as SEQ ID NOs: 1-5. Although the
precursor forms of miR-26a-1 and miR-16a-2 are different, the
sequences of the mature forms of these miRs are identical (SEQ ID
NO: 4).
[0133] Detection and quantification of microRNA expression can be
achieved by any one of a number of methods well known in the art
(see, for example, U.S. Patent Application Publication Nos.
2006/0211000 and 2007/0299030, herein incorporated by reference)
and described below. Using the known sequences for miR-26 family
members, specific probes and primers can be designed for use in the
detection methods described below as appropriate.
[0134] In some cases, the miRNA detection method requires isolation
of nucleic acid from a sample, such as a cell or tissue sample.
Nucleic acids, including RNA and specifically miRNA, can be
isolated using any suitable technique known in the art. For
example, phenol-based extraction is a common method for isolation
of RNA. Phenol-based reagents contain a combination of denaturants
and RNase inhibitors for cell and tissue disruption and subsequent
separation of RNA from contaminants. Phenol-based isolation
procedures can recover RNA species in the 10-200-nucleotide range
(e.g., precursor and mature miRNAs, 5S and 5.8S ribosomal RNA
(rRNA), and UI small nuclear RNA (snRNA)). In addition, extraction
procedures such as those using TRIZOL.TM. or TRI REAGENT.TM., will
purify all RNAs, large and small, and are efficient methods for
isolating total RNA from biological samples that contain miRNAs and
small interfering RNAs (siRNAs).
[0135] Microarray
[0136] A microarray is a microscopic, ordered array of nucleic
acids, proteins, small molecules, cells or other substances that
enables parallel analysis of complex biochemical samples. A DNA
microarray consists of different nucleic acid probes, known as
capture probes that are chemically attached to a solid substrate,
which can be a microchip, a glass slide or a microsphere-sized
bead. Microarrays can be used, for example, to measure the
expression levels of large numbers of messenger RNAs (mRNAs) and/or
miRNAs simultaneously.
[0137] Microarrays can be fabricated using a variety of
technologies, including printing with fine-pointed pins onto glass
slides, photolithography using pre-made masks, photolithography
using dynamic micromirror devices, ink-jet printing, or
electrochemistry on microelectrode arrays.
[0138] Microarray analysis of miRNAs can be accomplished according
to any method known in the art (see, for example, PCT Publication
No. WO 2008/054828; Ye et al., Nat. Med. 9(4):416-423, 2003; Calin
et al., N. Engl. J. Med. 353(17):1793-1801, 2005, each of which is
herein incorporated by reference). In one example, RNA is extracted
from a cell or tissue sample, the small RNAs (18-26-nucleotide
RNAs) are size-selected from total RNA using denaturing
polyacrylamide gel electrophoresis. Oligonucleotide linkers are
attached to the 5' and 3' ends of the small RNAs and the resulting
ligation products are used as templates for an RT-PCR reaction with
10 cycles of amplification. The sense strand PCR primer has a
fluorophore attached to its 5' end, thereby fluorescently labeling
the sense strand of the PCR product. The PCR product is denatured
and then hybridized to the microarray. A PCR product, referred to
as the target nucleic acid that is complementary to the
corresponding miRNA capture probe sequence on the array will
hybridize, via base pairing, to the spot at which the capture
probes are affixed. The spot will then fluoresce when excited using
a microarray laser scanner. The fluorescence intensity of each spot
is then evaluated in terms of the number of copies of a particular
miRNA, using a number of positive and negative controls and array
data normalization methods, which will result in assessment of the
level of expression of a particular miRNA.
[0139] In an alternative method, total RNA containing the small RNA
fraction (including the miRNA) extracted from a cell or tissue
sample is used directly without size-selection of small RNAs, and
3' end labeled using T4 RNA ligase and either a
fluorescently-labeled short RNA linker. The RNA samples are labeled
by incubation at 30.degree. C. for 2 hours followed by heat
inactivation of the T4 RNA ligase at 80.degree. C. for 5 minutes.
The fluorophore-labeled miRNAs complementary to the corresponding
miRNA capture probe sequences on the array will hybridize, via base
pairing, to the spot at which the capture probes are affixed. The
microarray scanning and data processing is carried out as described
above.
[0140] There are several types of microarrays than be employed,
including spotted oligonucleotide microarrays, pre-fabricated
oligonucleotide microarrays and spotted long oligonucleotide
arrays. In spotted oligonucleotide microarrays, the capture probes
are oligonucleotides complementary to miRNA sequences. This type of
array is typically hybridized with amplified PCR products of
size-selected small RNAs from two samples to be compared (such as
non-cancerous tissue and HCC liver tissue) that are labeled with
two different fluorophores. Alternatively, total RNA containing the
small RNA fraction (including the miRNAs) is extracted from the two
samples and used directly without size-selection of small RNAs, and
3' end labeled using T4 RNA ligase and short RNA linkers labeled
with two different fluorophores. The samples can be mixed and
hybridized to one single microarray that is then scanned, allowing
the visualization of up-regulated and down-regulated miRNA genes in
one assay.
[0141] In pre-fabricated oligonucleotide microarrays or
single-channel microarrays, the probes are designed to match the
sequences of known or predicted miRNAs. There are commercially
available designs that cover complete genomes (for example, from
Affymetrix or Agilent). These microarrays give estimations of the
absolute value of gene expression and therefore the comparison of
two conditions requires the use of two separate microarrays.
[0142] Spotted long Oligonucleotide Arrays are composed of 50 to
70-mer oligonucleotide capture probes, and are produced by either
ink-jet or robotic printing. Short Oligonucleotide Arrays are
composed of 20-25-mer oligonucleotide probes, and are produced by
photolithographic synthesis (Affymetrix) or by robotic
printing.
[0143] Quantitative RT-PCR
[0144] Quantitative RT-PCR (qRT-PCR) is a modification of
polymerase chain reaction used to rapidly measure the quantity of a
product of polymerase chain reaction. qRT-PCR is commonly used for
the purpose of determining whether a genetic sequence, such as a
miR, is present in a sample, and if it is present, the number of
copies in the sample. Any method of PCR that can determine the
expression of a nucleic acid molecule, including a miRNA, falls
within the scope of the present disclosure. There are several
variations of the qRT-PCR method known in the art, three of which
are described below.
[0145] Methods for quantitative polymerase chain reaction include,
but are not limited to, via agarose gel electrophoresis, the use of
SYBR Green (a double stranded DNA dye), and the use of a
fluorescent reporter probe. The latter two can be analyzed in
real-time.
[0146] With agarose gel electrophoresis, the unknown sample and a
known sample are prepared with a known concentration of a similarly
sized section of target DNA for amplification. Both reactions are
run for the same length of time in identical conditions (preferably
using the same primers, or at least primers of similar annealing
temperatures). Agarose gel electrophoresis is used to separate the
products of the reaction from their original DNA and spare primers.
The relative quantities of the known and unknown samples are
measured to determine the quantity of the unknown.
[0147] The use of SYBR Green dye is more accurate than the agarose
gel method, and can give results in real time. A DNA binding dye
binds all newly synthesized double stranded DNA and an increase in
fluorescence intensity is measured, thus allowing initial
concentrations to be determined. However, SYBR Green will label all
double-stranded DNA, including any unexpected PCR products as well
as primer dimers, leading to potential complications and artifacts.
The reaction is prepared as usual, with the addition of fluorescent
double-stranded DNA dye. The reaction is run, and the levels of
fluorescence are monitored (the dye only fluoresces when bound to
the double-stranded DNA). With reference to a standard sample or a
standard curve, the double-stranded DNA concentration in the PCR
can be determined.
[0148] The fluorescent reporter probe method uses a
sequence-specific nucleic acid based probe so as to only quantify
the probe sequence and not all double stranded DNA. It is commonly
carried out with DNA based probes with a fluorescent reporter and a
quencher held in adjacent positions (so-called dual-labeled
probes). The close proximity of the reporter to the quencher
prevents its fluorescence; it is only on the breakdown of the probe
that the fluorescence is detected. This process depends on the 5'
to 3' exonuclease activity of the polymerase involved.
[0149] The real-time quantitative PCR reaction is prepared with the
addition of the dual-labeled probe. On denaturation of the
double-stranded DNA template, the probe is able to bind to its
complementary sequence in the region of interest of the template
DNA. When the PCR reaction mixture is heated to activate the
polymerase, the polymerase starts synthesizing the complementary
strand to the primed single stranded template DNA. As the
polymerization continues, it reaches the probe bound to its
complementary sequence, which is then hydrolyzed due to the 5'-3'
exonuclease activity of the polymerase, thereby separating the
fluorescent reporter and the quencher molecules. This results in an
increase in fluorescence, which is detected. During thermal cycling
of the real-time PCR reaction, the increase in fluorescence, as
released from the hydrolyzed dual-labeled probe in each PCR cycle
is monitored, which allows accurate determination of the final, and
so initial, quantities of DNA.
[0150] In Situ Hybridization
[0151] In situ hybridization (ISH) applies and extrapolates the
technology of nucleic acid hybridization to the single cell level,
and, in combination with the art of cytochemistry,
immunocytochemistry and immunohistochemistry, permits the
maintenance of morphology and the identification of cellular
markers to be maintained and identified, and allows the
localization of sequences to specific cells within populations,
such as tissues and blood samples. ISH is a type of hybridization
that uses a complementary nucleic acid to localize one or more
specific nucleic acid sequences in a portion or section of tissue
(in situ), or, if the tissue is small enough, in the entire tissue
(whole mount ISH). RNA ISH can be used to assay expression patterns
in a tissue, such as the expression of miRNAs.
[0152] Sample cells or tissues are treated to increase their
permeability to allow a probe, such as a miRNA-specific probe, to
enter the cells. The probe is added to the treated cells, allowed
to hybridize at pertinent temperature, and excess probe is washed
away. A complementary probe is labeled with a radioactive,
fluorescent or antigenic tag, so that the probe's location and
quantity in the tissue can be determined using autoradiography,
fluorescence microscopy or immunoassay. The sample may be any
sample as herein described, such as a non-cancerous or HCC liver
sample. Since the sequences of miR-26 family members are known,
miR-26 probes can be designed accordingly such that the probes
specifically bind miR-26.
[0153] In Situ PCR
[0154] In situ PCR is the PCR based amplification of the target
nucleic acid sequences prior to ISH. For detection of RNA, an
intracellular reverse transcription step is introduced to generate
complementary DNA from RNA templates prior to in situ PCR. This
enables detection of low copy RNA sequences.
[0155] Prior to in situ PCR, cells or tissue samples are fixed and
permeabilized to preserve morphology and permit access of the PCR
reagents to the intracellular sequences to be amplified. PCR
amplification of target sequences is next performed either in
intact cells held in suspension or directly in cytocentrifuge
preparations or tissue sections on glass slides. In the former
approach, fixed cells suspended in the PCR reaction mixture are
thermally cycled using conventional thermal cyclers. After PCR, the
cells are cytocentrifuged onto glass slides with visualization of
intracellular PCR products by ISH or immunohistochemistry. In situ
PCR on glass slides is performed by overlaying the samples with the
PCR mixture under a coverslip which is then sealed to prevent
evaporation of the reaction mixture. Thermal cycling is achieved by
placing the glass slides either directly on top of the heating
block of a conventional or specially designed thermal cycler or by
using thermal cycling ovens.
[0156] Detection of intracellular PCR products is generally
achieved by one of two different techniques, indirect in situ PCR
by ISH with PCR-product specific probes, or direct in situ PCR
without ISH through direct detection of labeled nucleotides (such
as digoxigenin-11-dUTP, fluorescein-dUTP, 3H-CTP or
biotin-16-dUTP), which have been incorporated into the PCR products
during thermal cycling.
[0157] Use of miR-26 as a Predictive Marker of HCC Prognosis and
for Identification of Therapeutic Agents for Treatment of HCC
[0158] It is disclosed herein that miR-26 is an independent
predictor of survival prognosis in HCC patients. HCC tumor samples
with low miR-26 expression compared to non-cancerous tissue from
the same subject or from a healthy subject, predicts a decrease in
survival. In addition, when therapy outcomes from IFN-.alpha.
treatment of HCC patients were stratified, only patients with low
miR-26 expression in tumors responded to IFN-.alpha. therapy
favorably. Thus, miR-26 status in tumors can be used as a clinical
tool in HCC patients' prognosis and for selecting appropriate HCC
patients who can benefit from IFN-.alpha. adjuvant therapy to
prevent relapse. In some cases, IFN-.alpha. therapy is used after
radical liver resection.
[0159] In some embodiments, the expression level of miR-26 in a HCC
tumor sample is directly compared with the expression level of
miR-26 in surrounding non-cancerous tissue from the same patient.
In other embodiments, miR-26 expression in the tumor sample is
compared to the expression level of miR-26 in a liver sample
obtained from a healthy subject, such as a liver donor. In some
cases, the non-cancerous tissue used as a control sample is
obtained from a cadaver. In other embodiments, expression of miR-26
in the tumor sample is compared with a standard level based on
historical values. For example, the standard can be set based on
average expression levels of miR-26 in non-cancerous liver tissue
samples obtained from a cohort of subjects. For instance, the
cohort of subjects can be a group of HCC patients enrolled in a
clinical trial. The cohort of subject can also be a group of
cadaveric donors.
[0160] Low expression of one or more miR-26 family members in a HCC
tumor sample relative to a control indicates a poor prognosis for
the patient and identifies the patient as a good candidate for
IFN-.alpha. adjunctive therapy. As used herein, "poor prognosis"
generally refers to a decrease in survival, or in other words, an
increase in risk of death or a decrease in the time until death.
Poor prognosis can also refer to an increase in severity of the
disease, such as an increase in spread (metastasis) of the cancer
to other organs. In one embodiment, low expression of miR-26 is
indicated by at least a 1.5-fold decrease in expression relative to
the control. In other embodiments, low expression of miR-26 is
indicated by at least a 2-fold, at least a 2.5-fold, at least a
3-fold, at least a 3.5-fold, or at least a 4-fold decrease in
miR-26 expression relative to the control.
[0161] The finding that patients with HCC tumors having higher
levels of miR-26 expression have a better chance of survival
indicates that compounds that increase expression of miR-26 will be
useful as therapeutic agents for the treatment of HCC. Thus,
provided herein is a method of identifying therapeutic agents for
the treatment of HCC, comprising screening candidate agents in
vitro to select an agent that increases expression of miR-26 in HCC
cells. In some embodiments, screening comprises contacting the
candidate agents with HCC cells and detecting any change in the
expression level of miR-26 in the cells. The HCC cells can be
primary cells obtained from a HCC patient, immortalized or
transformed cells obtained from a patient, or the cells can be
commercially available immortalized cell lines, such as, but not
limited to MHCC97, HepG2, Hep3B or SNU-423 cells.
[0162] An increase in expression of miR-26 following treatment with
the candidate agent identifies the agent as a therapeutic agent for
the treatment of HCC. Methods of screening candidate agents to
identify therapeutic agents for the treatment of disease are well
known in the art. Methods of detecting expression levels of miR-26
are known in the art and are described herein, such as, but not
limited to, microarray analysis, RT-PCR (including qRT-PCR), in
situ hybridization, in situ PCR, and Northern blot analysis. In one
embodiment, screening comprises a high-throughput screen. In
another embodiment, candidate agents are screened individually.
[0163] The candidate agents can be any type of molecule, such as,
but not limited to nucleic acid molecules, proteins, peptides,
antibodies, lipids, small molecules, chemicals, cytokines,
chemokines, hormones, or any other type of molecule that may alter
miR-26 expression either directly or indirectly. In some
embodiments, the candidate agents are molecules that play a role in
the NF.kappa.B/IL-6 signaling pathway. In other embodiments, the
candidate agents are molecules that play a role in the IL-10, STAT3
or interferon-inducible factor signaling networks. In one
embodiment, the candidate agents are cytokines. In another
embodiment, the candidate agents are small molecules.
[0164] Also described herein is a method for the characterization
of hepatocellular carcinoma (HCC), wherein at least one feature of
HCC is selected from one or more of the group consisting of:
presence or absence of HCC; diagnosis of HCC; prognosis of HCC;
therapy outcome prediction; therapy outcome monitoring; suitability
of HCC to treatment, such as suitability of HCC to chemotherapy
treatment and/or radiotherapy treatment; suitability of HCC to
hormone treatment; suitability of HCC for removal by invasive
surgery; suitability of HCC to combined adjuvant therapy.
[0165] Also described herein is a kit for the detection of HCC, the
kit comprising at least one detection probe comprising one or more
members of the miR-26 family. The kit can be in the form or
comprises an oligonucleotide array.
[0166] Also described herein is a method for the determination of
suitability of a HCC patient for treatment comprising: i) isolating
at least one tissue sample from a patient suffering from HCC;
[0167] ii) performing the characterization of at least one tissue
sample and/or utilizing a detection probe, to identify at least one
feature of the HCC; iii) based on the at least one feature
identified in step ii), diagnosing the physiological status of the
patient; iv) based on the diagnosis obtained in step iii),
determining whether the patient would benefit from treatment of the
HCC.
[0168] In certain embodiments, the at least one feature of the
cancer is selected from one or more of the group consisting of:
presence or absence of the cancer; type of the cancer; origin of
the cancer; diagnosis of cancer; prognosis of the cancer; therapy
outcome prediction; therapy outcome monitoring; suitability of the
cancer to treatment, such as suitability of the cancer to
chemotherapy treatment and/or radiotherapy treatment; suitability
of the cancer to hormone treatment; suitability of the cancer for
removal by invasive surgery; suitability of the cancer to combined
adjuvant therapy.
[0169] Also described herein is a method of for the determination
of suitability of a cancer for treatment, wherein the at least one
feature of the cancer is suitability of the cancer to treatment,
such as suitability of the cancer to chemotherapy treatment and/or
radiotherapy treatment; suitability of the cancer to hormone
treatment; suitability of the cancer for removal by invasive
surgery; suitability of the cancer to combined adjuvant
therapy.
[0170] Also described herein is a method for the determination of
the likely prognosis of a HCC patient comprising: i) isolating at
least one tissue sample from a patient suffering from HCC; and, ii)
characterizing at least one tissue sample to identify at least one
feature of the HCC; wherein the feature allows for the
determination of the likely prognosis of the HCC patient.
[0171] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
[0172] The Examples below describe the analysis of gender-dependent
microRNA profiles in HCC and their predictive values in survival
prognosis and therapeutic outcomes. For these studies, two
independent cohorts of 379 total HCC patients were analyzed. The
first cohort was a test cohort to identify potential microRNAs
associated with HCC. The second cohort (the validation cohort) was
used to confirm the results obtained from the test cohort. Using
this strategy, members of miR-26 were identified as gender-related
microRNAs as they were more abundantly expressed in female hepatic
tissues. In addition, the expression levels of miR-26 family
members were significantly downregulated in a subset of HCC tumor
samples as compared to their paired non-cancerous tissues
regardless of gender. Tumors with reduced miR-26 expression had a
distinct gene expression profile, and cases with low miR-26
expression were associated with poor survival prognosis. The data
described below suggests that miR-26 functions as a tumor
suppressor and tumors with miR-26 silencing may be biologically
unique.
Example I
Materials, Methods and Patient Characteristics
[0173] Clinical Specimens
[0174] Snap frozen or paraffin-embedded specimens of both tumors
(T) and surrounding non-tumor hepatic tissues (NT) were obtained
with informed consent from 455 HCC patients who underwent radical
resection at the Liver Cancer Institute of Fudan University,
Shanghai (376 cases) and at the University of Hong Kong Medical
Centre, Hong Kong (79 cases), China (4). The study was approved by
the Institutional Review Board of the corresponding institutes. A
normal liver tissue sample pool was obtained from 8 disease-free
liver donors (24). A previously described cohort of 241 HCC cases
(cohort 1: test cohort), with available microRNA microarray data
(22), was used to search for microRNAs associated with gender and
survival. Among them, 17 had missing miR-26 expression data and 9
had missing survival data, leaving 224 cases for miR-26 expression
analysis and 217 cases for survival analyses. HCC cases (n=135)
from prospective randomized control trials (RCT) (cohort 2:
validation cohort and IFN test cohort) (3) to evaluate adjuvant IFN
therapy were used as an independent validation cohort by
quantitative reverse transcription polymerase chain reaction
(qRT-PCR). Among cohort 2, 6 had missing miR-26 expression data and
12 had missing survival data, leaving 129 cases (60 controls, 69
IFN cases) for miR-26 expression analysis and 118 cases (59
controls, 59 IFN cases) for survival analysis. The remaining 79 HCC
cases (40 controls, 39 IFN cases) from another prospective RCT
(cohort 3: IFN validation cohort) were used to validate the
association between miR-26 and interferon therapy (4). Additional
methodology is described in detail in the Supplementary
Appendix.
[0175] Characteristics of the Patients
[0176] The study involved two independent cohorts consisting of 376
patients from the Liver Cancer Institute in Shanghai (Table 1) and
an additional IFN validation cohort (FIG. 4--Supplemental Table 1)
from the University of Hong Kong, all with histologically-confirmed
HCC and a majority (90.5%) of hepatitis B virus (HBV) chronic
carriers. Cohort 1 consisted of 241 patients, while cohort 2
consisted of 135 patients who participated in a prospective RCT of
interferon therapy (3). Cohort 3 consisted of 79 HCC patients from
another prospective RCT of interferon therapy (4). All patients
received liver resections with curative intent. A majority of the
patients were males (85.1%), with cirrhosis (88.1%), elevated serum
AFP (62.2%) and a solitary presentation (84.4%). Clinical variables
were similar between test and validation cohorts, with the
exception of serum alanine transaminase (ALT) level, TNM staging
and adjuvant therapy. Liver inflammation activity in these
HBV-related HCC patients, as indicated by ALT levels, was
significantly lower in cohort 2 cases than cohort 1 or cohort 3 and
more early stage HCC cases were found in cohort 2. In addition, 39
patients in cohort 1 received prophylactic adjuvant therapies, but
the responses appeared minimum (p=0.9; log rank test). In contrast,
53.3% of patients in cohort 2 and 49.4% in cohort 3 received an
`intent-to-cure` IFN adjuvant therapy, which improved overall
survival (3; 4).
[0177] Gender-Related microRNAs and Clinical Outcome
[0178] To search for differentially expressed microRNAs between
male and female liver samples, we globally analyzed the microRNA
expression profiles of 241 cases from cohort 1, where both tumor
(T) and non-tumor (NT) microRNA array data were available (GEO
accession number, GSE6857) (22). To avoid potential confounding
factors, an age-matched and balanced case set was used to identify
gender-dependent microRNAs, which contained all female cases (n=30)
and two age-matched male groups, i.e., G1 (n=31) and G2 (n=31). The
clinical characteristics of female cases and male G1 or G2 cases
were similar (FIG. 4C--Supplemental Table 2).
[0179] Class comparison analysis revealed that 15 (female vs. male
G1) or 45 (female vs. male G2) microRNAs were differentially
expressed in NT tissues, while 7 overlapped. In contrast, only one
overlapping microRNA, miR-129-2, was found in tumors (FIG.
6A--Supplemental Table 3). Therefore, there were more consistent
differences in microRNA expression in hepatic microenvironments
than in tumors.
[0180] Among the overlapping microRNAs, miR-26a-1 was chosen for
further analysis since its level was most significantly different
between genders and was most abundant. Analysis with cohort 1 cases
showed that miR-26a-1 level was significantly higher in female
livers than males (FIG. 1A).
[0181] This was validated by mature miR-26 expression in female
cases (n=26) and age-matched male cases (n=56) using qRT-PCR (FIG.
1B).
[0182] The inventors herein then reasoned that miR-26 may act as a
gender-dependent tumor suppressor gene and if so, silencing of
miR-26 would be a frequent event in tumors. Analyses showed that a
significant reduction of miR-26a-1 in T compared to NT samples was
observed in only low miR-26 cases (p<0.001) but not in high
miR-26 cases (p=0.23) (FIG. 1C) when 224 HCC cases were
dichotomized (low or high miR-26 based on the median level of
miR-26a-1 in tumors).
[0183] The median fold change (T/NT ratio) was 0.37 in low
miR-26a-1 cases and 0.98 in high miR-26a-1 cases, suggesting that
silencing of miR-26 was only associated with low miR-26 cases.
Moreover, the low miR-26 cases were associated with poor survival
(FIG. 1D).
[0184] In humans, there are three miR-26 members, i.e., miR-26a-1,
miR-26a-2 and miR-26b. These microRNAs are evolutionarily highly
conserved with 26a-1 and 26a-2 sharing an identical mature
sequence, suggesting their functional redundancy. The expression
patterns of all three miR-26 members and their associations with
survival were similar (FIG. 7).
[0185] The inventors herein now show that miR-26 members were more
abundantly expressed in female livers and their silencing may be
important in the development of a subset of HCCs with poor
outcome.
[0186] Distinct Gene Expression Patterns are Associated with Low
miR-26 HCCs
[0187] To test whether that the low miR-26 HCCs may be biologically
distinct, the inventors herein analyzed 224 matched HCC cases with
available microRNA and mRNA microarray data. The mRNA microarray
data were based on the expression of .about.21,000 mRNA genes (GEO
accession number, GSE5975) (27). Multidimensional scaling analysis,
based on the first three principal components of all genes,
revealed that a majority of low miR-26 cases clustered separately
from high miR-26 cases (FIG. 2A), according to the dichotomized
expression status of the three miR-26s (FIG. 8).
[0188] Class comparison analysis showed that the expression of a
significant number of genes differed in tumors between low and high
miR-26s groups and 915 genes were in common (FIG. 2B).
[0189] SLC2A6 and S100P were selected among the differentially
expressed genes for validation by qRT-PCR (FIG. 9).
[0190] Further, a multivariate class prediction analysis resulted
in a significant class prediction of low miR-26 cases with 80.3%
overall accuracy. Thus, low miR-26 HCC cases are distinct in their
gene expression patterns compared to high miR-26 HCC cases.
[0191] Among the 915 overlapping genes, 770 were overexpressed in
low miR-26 HCCs. Gene network analyses using these 770 genes
revealed a series of putative tumorigenesis-networks with a high
score (>10) (FIG. 6B--Table 4).
[0192] Examination of the enriched genes in various categories
revealed several significant signaling networks, the most striking
of which showed a predominant activation of the NF.kappa.B/IL-6
signaling pathway in low miR-26 cases (FIG. 2C).
[0193] We measured the level of the NF.kappa.B target gene, IL-6,
by qRT-PCR, as it was related to HCC and HCC gender disparity. Most
of the HCC cases with a reduced miR-26 level had a concomitant
elevation of IL-6 expression (FIG. 10). Taken together, these data
show that low miR-26 HCCs have a distinct gene profile.
[0194] Validation with Independent Cohorts
[0195] To validate the association of gender-dependent miR-26s with
survival, we detected mature miR-26s by qRT-PCR in T and NT tissues
from cohort 2. As IFN adjuvant therapy altered survival outcome, we
analyzed the control group. Consistent with cohort 1, miR-26
expression was more abundant in female NT tissues but a significant
reduction was observed in tumors, regardless of gender (FIG.
11).
[0196] Moreover, low miR-26 expression in tumors was significantly
associated with poor patient survival (FIG. 3A, FIG. 12A).
[0197] Another independent cohort (cohort 3) showed consistent
results (FIG. 3B, FIG. 12B).
[0198] Cox proportional hazards regression analysis was used to
further evaluate the association of miR-26 expression with
prognosis in controls among cohort 2 (Table 2).
[0199] In univariate analysis, low miR-26a tumor expression and TNM
staging were significantly associated with prognosis. The final
multivariate model revealed that low miR-26a expression in tumors
was an independent predictor of poor survival. A similar trend was
found for miR-26b. Thus, the dichotomized miR-26 expression values
were independent predictors of prognosis.
[0200] miR-26 Expression and Therapeutic Outcome
[0201] Low miR-26 HCCs appeared to be biologically distinct with an
enrichment of genes functionally linked to immunobiology including
those in the NF.kappa.B/IL-6 pathway. While not wishing to be bound
by theory, the inventors herein now believe that such a tumor may
be `addictive` in its response to cytokine-mediated activity. Since
cohort 2 consisted of cases treated with IFN, the inventors
analyzed associations between miR-26 expression and therapeutic
outcome.
[0202] Patients with low miR-26a expression in tumors had a
significantly improved overall survival after receiving IFN
adjuvant therapy when compared to those in the control group
(p=0.003) (FIG. 3C), which was validated in cohort 3 (FIG. 3D).
[0203] In contrast, patients with high miR-26a expression from both
cohorts did not respond to IFN (FIGS. 3E-3F).
[0204] Similar results were obtained with miR-26b expression (FIG.
12C-12F). Cox proportional hazards regression analysis was used to
evaluate the effect of treatment on survival in low miR-26 groups
of cohort 2 (FIG. 5B--Table 3).
[0205] In both univariate and multivariate analyses, IFN treatment
was associated with a significantly improved survival in the low
miR-26 group. An interaction analysis between miR-26 expression,
IFN treatment, and survival also showed that miR-26 expression
significantly affects IFN-associated survival outcome in these two
cohorts (miR-26a, p=0.004; miR-26b, p=0.02). Thus, miR-26 is an
independent predictor of IFN response.
[0206] Discussion
[0207] This is the largest study to date analyzing gender-dependent
microRNA profiles in HBV-related HCCs and their predictive values
in survival prognosis and therapeutic outcomes using three
independent cohorts.
[0208] The inventors herein have not shown that miR-26s were more
abundantly expressed in female hepatic tissues, but their
expression was significantly downregulated in a subset of HCCs
compared to their paired non-cancerous tissues regardless of
gender. These results indicate that miR-26s are gender and
tumor-related microRNAs.
[0209] Also, tumors with reduced miR-26 expression had a distinct
gene expression profile, and cases with low miR-26 expression had
poor prognosis but responded favorably to IFN therapy.
[0210] These results show that miR-26 may be a tumor suppressor.
miR-26 silencing in hepatocytes can contribute to male predominance
in the development of an aggressive HCC. The following findings are
consistent with the inventors' hypothesis above: (1) miR-26 is
expressed at higher levels in female livers, where presumably more
anti-carcinogenic activities exist; (2) miR-26 expression is
silenced in a subset of HCCs with poor survival; (3) Genes
activated in low miR-26 HCCs are selectively enriched in
NF.kappa.B/IL-6 signaling pathways. (4) miR-26 is expressed more
abundantly in hepatocytes and immortalized/non-transformed
hepatocytes than in HCC cells and PBMC (FIG. 13).
[0211] Gender disparity of liver cancer was recently found to be
due to gender differences in MyD8-dependent IL-6 induction by
NF-.kappa.B in mice (13). Intriguingly, estrogens inhibit IL-6
promoter activity, which may contribute to a decreased
susceptibility to HCC in females.
[0212] These are consistent with these findings as IL-6 expression
was inversely correlated with miR-26. Moreover, many genes
activated in low miR-26 HCCs can function in inducing progesterone,
but inhibit estrogen signaling (networks 5 and 15, FIG.
6B--Supplemental Table 4). Interestingly, miR-26 expression was not
associated with hepatic growth factor (HGF), another important
factor in HCC (FIG. 14).
[0213] The inventors' herein analyses revealed that miR-26 was an
independent predictor of survival. However, when therapy outcomes
from IFN treatment were stratified, only patients with low miR-26
expression in tumors responded favorably to IFN therapy in two
independent prospective RCTs.
[0214] These results indicate that miR-26 status in tumors is a
useful clinical tool in HCC patient prognosis and in assisting
selection of appropriate HCC patients who can benefit from IFN
adjuvant therapy to prevent relapse.
[0215] Currently, recurrence-related mortality is a significant
clinical problem for HCC patients who receive surgery and a single
agent is not available as standard care at an adjuvant setting.
Encouragingly, Clavien evaluated the adjuvant effects of IFN after
liver resection or tumor ablation in 7 RCTs and concluded that all
of these trials showed modest beneficial effects, but an
improvement is clearly needed (5). In addition, among multiple
experimental agents, only a modest survival benefit is observed
with sorafenib (6). The poor efficacy of current systemic
therapeutic agents may be caused by an inability to select a
subpopulation of patients who may respond most favorably to a
particular HCC therapy.
[0216] For the first time, these results provide a solution to this
problem. The results described herein now have led to a rediscovery
of a `historical` agent, i.e., IFN, whose traditional modest
therapeutic benefit may now be shifted to an agent with great
potential.
[0217] Due to the robustness of the miR-26 predictor, IFN can be
used as a first line therapy for HCC patients who receive resection
and have tumors with reduced miR-26 expression, which will need to
be evaluated in prospective studies. It should be noted that the
studies presented here were mainly from HBV-positive (.about.90%)
Chinese HCC patients, and will therefore need to be evaluated in
non-Asian HCCs and HCCs arising from other underlying liver
diseases such as hepatitis C and/or alcohol.
[0218] While the mechanism(s) behind the sensitivity of low miR-26
HCC cases to IFN treatment is currently unclear, the inventors
herein believe that these HCCs represent a unique type of tumor
with a specific activation of the IFN-responsive signaling pathway.
Consistently, low miR-26 HCCs were distinct from high miR-26 HCCs
and had poor survival prognosis. Many of the overexpressing genes
in low miR-26 HCCs are related to cell immunity such as those
encoding pro- and anti-inflammatory cytokines (i.e., IL-1, IL-2,
IL-10 and IL-17).
[0219] Moreover, many signaling networks activated in low miR-26
HCCs are immune-associated such as NF.kappa.B/IL-6, IL-10, STAT3
and IFN-inducible factor signaling networks.
[0220] Again, while not wishing to be bound by theory, the
inventors herein believe that tumors with low miR-26 expression may
have a unique activation of IFN signaling, potentially through
NF.kappa.B/IL-6 signaling pathway, and thus may be sensitive to
IFN-mediated growth inhibition via IL-6/STAT3 signaling (29).
[0221] Now described herein is the identification of systematic
differences in microRNA expression patterns between male and female
liver tissues derived from HCC patients. Tumors with a reduced
miR-26 expression were biologically distinct, had poor survival
outcome, but responded favorably to adjuvant IFN therapy. These
data indicate that miR-26 is a useful diagnostic and prognostic
biomarker for HCC and can assist in selecting patients who can
significantly benefit from adjuvant IFN therapy.
Example II
RNA Isolation and Real-Time qRT-PCR Analysis
[0222] Total RNAs were extracted from frozen tissues of cohort 1
using standard TRIZOL (Invitrogen, Carlsbad, Calif.) methods, and
from paraffin-embedded tissues of cohort 2 and cohort 3 using a
MasterPure RNA Purification Kit (Epicenter, Madison, Wis.). The
expression of mature microRNAs was measured using Taqman MicroRNA
Assays specific for miR-26a and miR-26b after reverse transcription
(Applied biosystems, Foster City, Calif.). All comparisons between
strata (gender. miR etc) were within each cohort. The Taqman
MicroRNA Assay for U6 RNA was used to normalize the relative
abundance of microRNAs. The expression of IL-6 was measured using
the Taqman Gene Assay specific for this gene after reverse
transcription by using the High Capacity cDNA Archive Kit (Applied
Biosystems, Foster City, Calif.). The Taqman gene assay for 18s was
used to normalize the relative abundance of mRNA. The experiments
were performed in triplicate.
[0223] Microarray Analyses and Statistics
[0224] For microRNA microarray profiling, tumors and paired
non-tumor tissues were profiled separately using a single channel
array platform previously described (22). The array quality
control, data preprocessing and normalization were done essentially
as previously described (22). The BRB-ArrayTools software 3.6.0
(http://linus.nci.nih.gov) was used for microarray analyses as
previously described (25; 26). MicroRNA probes with values missing
from more than 50% of the arrays and those with less than 20% of
expression data values having at least a 1.5-fold change in either
direction from the probe's median value were excluded from the
analysis, which left 624 probes. Class comparison analysis using
t-statistics was used to identify microRNAs that were
differentially expressed in tumors or surrounding non-cancerous
tissues between males and females.
[0225] For this analysis, the initial significance threshold of
univariate tests was set at p<0.05 and the analyses were based
on 1000 permutations for the multivariate test to generate
permutation p-values for the global test to control false discovery
rates. For mRNA expression microarray profiling, the inventors used
their our previously available oligoarray dataset based on a
dual-channel platform (i.e., T/NT ratio) (27) that contained 224
cases matched to those with available microRNA microarray data
described above. The inventors used the median expression in tumors
to dichotomize HCC cases, where low miR-26 expression was
classified as the lower 50th percentile and high miR-26 expression
was classified as the upper 50th percentile. Class comparison
analysis based on dichotomized miR-26 expression levels was used to
identify differentially expressed mRNAs between low miR-26 and high
miR-26 HCCs. The same probe filtering criteria was followed as
described above, leaving 11,580 expression probes for these
comparisons.
[0226] Six class prediction algorithms, i.e., Support Vector
Machines (SVM), Compound Covariate Predictor (CCP), Diagonal Linear
Discriminant (DLD), 1-Nearest Neighbor (1NN), 3-Nearest Neighbor
(3NN) or Nearest Centroid (NC), were also used to determine whether
mRNA expression patterns could accurately discriminate low miR-26
HCCs from high miR-26 HCCs. In these analyses, 90% of the samples
were randomly chosen to build a classifier which was then used to
predict the remaining 10% of the cases. The accuracy of the
prediction was calculated after 1000 repetitions of this random
partitioning process to control the number and proportion of false
discoveries. Hierarchical clustering analysis was performed using
BRBarrayTools with median-centered correlation and complete
linkage. Using an unsupervised approach, we also performed
multidimensional scaling analysis using all cohort 1 samples based
on the first three principal components of 11,580 genes that passed
the filter. The expression levels of these genes were
log-transformed, and Euclidean distance was used to determine their
positions. Gene Network Analyses were used to identify signaling
pathways that were enriched with genes differentially expressed in
tumors between miR-26 low and miR-26 high HCCs using Ingenuity
Pathways Analysis (Ingenuity.RTM., www.ingenuity.com).
[0227] Kaplan-Meier survival analysis was used to compare patient
survival based on dichotomized miR-26 expression, using GraphPad
Prism software 5.0 (GraphPad Software, San Diego, Calif.) with
statistical P values generated by the Cox-Mantel log-rank test. Cox
proportional hazards regression analyses were used to analyze the
effect of clinical variables on patient survival using STATA 9.2
(College Station, Tex.). A univariate test was used to examine the
influence of each clinical variable on survival. A multivariate
analysis was performed considering clinical variables from the
univariate analysis that were significantly associated with
survival with significance set at p<0.05. Multi-colinearity of
the covariates was assessed and was not found to be present. In the
final models, gender was included as a covariate due to its
biological relevance in HCC outcome and its association with miR-26
expression. It was determined that the final models met the
proportional hazards assumption. For RT-PCR data, the statistical P
value, generated by the student t-test, and the Spearman
correlation constant were calculated using GraphPad Prism Software
5.0. The statistical significance was defined as p<0.05. All
p-values in this paper are two-sided.
Example III
Method of Treating HCC in Patients Exhibiting Low Expression of
miR-26 in HCC Tumor Samples
[0228] This example describes a method of selecting and treating
HCC patients that are likely to have a favorable response to
IFN-.alpha. treatment as an adjunctive therapy.
[0229] For some HCC patients, adjuvant therapies, such as
IFN-.alpha. therapy can prolong survival (Sun et al., J. Cancer
Res. Clin. Oncol. 132(7):458-465, 2006). However, it would be
beneficial to identify patients that are most likely to benefit
from IFN-.alpha. adjunctive therapy prior to initiating
treatment.
[0230] It is now disclosed herein that the prognosis of HCC
patients expressing low levels of miR-26 in HCC tumor samples
relative to a control (such as non-cancerous liver tissue obtained
from the same patient) significantly improves after treatment with
IFN-.alpha.. In contrast, patients expressing high levels of miR-26
in tumor samples do not exhibit a significant increase in survival
following IFN-.alpha. treatment and thus are not good candidates
for such adjunctive treatment.
[0231] A patient diagnosed with HCC first undergoes liver resection
with an intent to cure. HCC tumor and non-cancerous tissue samples
are obtained from the portion of the liver tissue removed from the
patient. RNA is then isolated from the tissue samples using any
appropriate method for extraction of small RNAs that are well known
in the art, such as by using TRIZOL.TM.. Purified RNA is then
subjected to RT-PCR using primers specific for miR-26 to determine
the expression level of miR-26 in the tumor and non-cancerous
tissues. If expression of miR-26 is at least 1.5-fold lower in the
tumor tissue relative to the non-cancerous tissue, the patient is a
candidate for IFN-.alpha. adjunctive therapy.
[0232] Accordingly, the patient is treated with a therapeutically
effective amount of IFN-.alpha. according to methods known in the
art (see, for example, Sun et al., J. Cancer Res. Clin. Oncol.
132(7):458-465, 2006; Qian et al., Cancer 107(7):1562-1569, 2006,
both of which are herein incorporated by reference). The dose and
dosing regimen of IFN-.alpha. will vary depending on a variety of
factors, such as health status of the patient and the stage of the
HCC. Typically, IFN-.alpha. is administered 1-3 times per week for
up to about six months.
Example IV
Alternative Treatment Method for HCC Patients with Low Expression
of miR-26
[0233] This example describes a method of treating a patient
diagnosed with HCC and exhibiting low expression of miR-26 with
interferon therapy in the absence of liver resection. To determine
whether a patient diagnosed with HCC is a good candidate for
IFN-.alpha. therapy, a HCC tumor sample is obtained from the
patient that has not undergone liver resection, along with a
non-cancerous liver tissue sample. The tissue samples can be
obtained according to any method known in the art. For example, the
tissue samples can be obtained by performing a biopsy procedure
using a hypodermic needle to remove the desired tissues.
[0234] RNA is then isolated from the tissue samples using any
appropriate method for extraction of small RNAs that are well known
in the art, such as by using TRIZOL.TM.. Purified RNA is then
subjected to RT-PCR using primers specific for miR-26 to determine
the expression level of miR-26 in the tumor and non-cancerous
tissues. If expression of miR-26 is at least 1.5-fold lower in the
tumor tissue relative to the non-cancerous tissue, the patient is a
candidate for IFN-.alpha. therapy.
[0235] Accordingly, the patient is treated with a therapeutically
effective amount of IFN-.alpha. according to methods known in the
art (see, for example, Sun et al., J. Cancer Res. Clin. Oncol.
132(7):458-465, 2006; Qian et al., Cancer 107(7):1562-1569, 2006,
both of which are herein incorporated by reference). The dose and
dosing regimen of IFN-.alpha. will vary depending on a variety of
factors, such as health status of the patient and the stage of the
HCC. Typically, IFN-.alpha. is administered 1-3 times per week for
up to about six months.
Example V
Method of Treating HCC in Patients Exhibiting High Expression of
miR-26 in HCC Tumor Samples
[0236] This example describes a method of treating a patient
diagnosed with HCC if the patient exhibits a high level of
expression of miR-26 in the HCC tumor.
[0237] A patient diagnosed with HCC first undergoes liver resection
with an intent to cure. HCC tumor and non-cancerous tissue samples
are obtained from the portion of the liver tissue removed from the
patient. RNA is then isolated from the tissue samples using any
appropriate method for extraction of small RNAs that are well known
in the art, such as by using TRIZOL.TM.. Purified RNA is then
subjected to RT-PCR using primers specific for miR-26 to determine
the expression level of miR-26 in the tumor and non-cancerous
tissues. If expression of miR-26 is not at least 1.5-fold lower in
the tumor tissue relative to the non-cancerous tissue, the patient
is unlikely to respond favorably to IFN-.alpha. adjunctive therapy.
Accordingly, the patient does not receive IFN-.alpha. therapy but
is monitored for post-operative signs of disease recurrence.
Example VI
Methods of Diagnosing HCC Patients
[0238] In one particular aspect, there is provided herein a method
of diagnosing whether a subject has, or is at risk for developing,
hepatocellular carcinoma (HCC). The method generally includes
measuring the level of at least one miR gene product in a test
sample from the subject and determining whether an alteration in
the level 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, HCC. In certain embodiments, the level of the at least
one miR gene product is measured using Northern blot analysis.
Also, in certain embodiments, the level of the at least one miR
gene product in the test sample is less than the level of the
corresponding miR gene product in the control sample, and/or the
level of the at least one miR gene product in the test sample is
greater than the level of the corresponding miR gene product in the
control sample.
Example VII
Measuring miR Gene Products
[0239] The level of the at least one miR gene product can be
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 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. An alteration in the signal of at least one
miRNA is indicative of the subject either having, or being at risk
for developing, HCC.
Example VIII
Diagnostic and Therapeutic Applications
[0240] In another aspect, there is provided herein are methods of
treating HCC in a subject, where the signal of at least one miRNA,
relative to the signal generated from the control sample, is
de-regulated (e.g., down-regulated and/or up-regulated).
[0241] Also provided herein are methods of diagnosing whether a
subject has, or is at risk for developing, a HCC associated with
one or more adverse prognostic markers in a subject, by 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. An alteration in the signal is indicative of the
subject either having, or being at risk for developing, the
cancer.
[0242] Also provided herein are methods of treating HCC in a
subject who has HCC in which at least one miR gene product is
down-regulated or up-regulated in the cancer cells of the subject
relative to control cells. When the one or more miR gene product is
down-regulated in the cancer cells, the method comprises
administering to the subject an effective amount of at least one
isolated miR gene product, such that proliferation of cancer cells
in the subject is inhibited. When one or more 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 at least one miR gene
product, such that proliferation of cancer cells in the subject is
inhibited.
[0243] Also provided herein are methods of treating HCC in a
subject, comprising: determining the amount of at least one miR
gene product in HCC cells, relative to control cells; and, altering
the amount of miR gene product expressed in the HCC cells by:
administering to the subject an effective amount of at least one
isolated miR gene product, if the amount of the miR gene product
expressed in the cancer cells is less than the amount of the miR
gene product expressed in control cells; or administering to the
subject an effective amount of at least one compound for inhibiting
expression of the at least one miR gene product, if the amount of
the miR gene product expressed in the cancer cells is greater than
the amount of the miR gene product expressed in control cells, such
that proliferation of cancer cells in the subject is inhibited.
Example IX
Compositions
[0244] Also provided herein are pharmaceutical compositions for
treating HCC, comprising at least one isolated miR gene product and
a pharmaceutically-acceptable carrier. In a particular embodiment,
the pharmaceutical compositions comprise at least one isolated miR
gene product corresponds to a miR gene product that is
down-regulated in HCC cells relative to suitable control cells. In
certain embodiments, the miR gene product comprises one or more of
the SEQ ID NOS: 1-5.
[0245] In another particular embodiment, the pharmaceutical
composition comprises at least one miR expression regulator (for
example, an inhibitor) compound and a pharmaceutically-acceptable
carrier.
[0246] Also provided herein are pharmaceutical compositions that
include at least one miR expression regulator compound that is
specific for a miR gene product that is up- or down-regulated in
HCC cells relative to suitable control cells.
[0247] Also provided herein are methods of identifying an anti-HCC
agent, comprising providing a test agent to a cell and measuring
the level of at least one miR gene product associated with
decreased expression levels in HCC cells, wherein 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
anti-HCC agent. In certain embodiments, the miR gene product
comprises one or more of the SEQ ID NOS: 1-5.
[0248] Also provided herein are methods of identifying an anti-HCC
agent, comprising providing a test agent to a cell and measuring
the level of at least one miR gene product associated with
increased expression levels in HCC cells, wherein 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 anti-HCC
agent.
Example X
Kits
[0249] Any of the compositions described herein may be comprised in
a kit. In a non-limiting example, reagents for isolating miRNA,
labeling miRNA, and/or evaluating an miRNA population using an
array are included in a kit. The kit may further include reagents
for creating or synthesizing miRNA probes. The kits will thus
comprise, in suitable container means, an enzyme for labeling the
miRNA by incorporating labeled nucleotide or unlabeled nucleotides
that are subsequently labeled. It may also include one or more
buffers, such as reaction buffer, labeling buffer, washing buffer,
or a hybridization buffer, compounds for preparing the miRNA
probes, and components for isolating miRNA. Other kits may include
components for making a nucleic acid array comprising
oligonucleotides complementary to miRNAs, and thus, may include,
for example, a solid support.
[0250] For any kit embodiment, including an array, there can be
nucleic acid molecules that contain a sequence that is identical or
complementary to all or part of any of SEQ ID NOS: 1-5.
[0251] The components of the kits may be packaged either in aqueous
media or in lyophilized form. The container means of the kits will
generally include at least one vial, test tube, flask, bottle,
syringe or other container means, into which a component may be
placed, and preferably, suitably aliquoted. Where there is more
than one component in the kit (labeling reagent and label may be
packaged together), the kit also will generally contain a second,
third or other additional container into which the additional
components may be separately placed. However, various combinations
of components may be comprised in a vial. The kits of the present
invention also will typically include a means for containing the
nucleic acids, and any other reagent containers in close
confinement for commercial sale. Such containers may include
injection or blow-molded plastic containers into which the desired
vials are retained.
[0252] When the components of the kit are provided in one and/or
more liquid solutions, the liquid solution is an aqueous solution,
with a sterile aqueous solution being one preferred solution. Other
solutions that may be included in a kit are those solutions
involved in isolating and/or enriching miRNA from a mixed
sample.
[0253] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means. The kits may also include
components that facilitate isolation of the labeled miRNA. It may
also include components that preserve or maintain the miRNA or that
protect against its degradation. The components may be RNAse-free
or protect against RNAses.
[0254] Also, the kits can generally comprise, in suitable means,
distinct containers for each individual reagent or solution. The
kit can also include instructions for employing the kit components
as well the use of any other reagent not included in the kit.
Instructions may include variations that can be implemented. It is
contemplated that such reagents are embodiments of kits of the
invention. Also, the kits are not limited to the particular items
identified above and may include any reagent used for the
manipulation or characterization of miRNA.
[0255] It is also contemplated that any embodiment discussed in the
context of an miRNA array may be employed more generally in
screening or profiling methods or kits of the invention. In other
words, any embodiments describing what may be included in a
particular array can be practiced in the context of miRNA profiling
more generally and need not involve an array per se.
[0256] It is also contemplated that any kit, array or other
detection technique or tool, or any method can involve profiling
for any of these miRNAs. Also, it is contemplated that any
embodiment discussed in the context of an miRNA array can be
implemented with or without the array format in methods of the
invention; in other words, any miRNA in an miRNA array may be
screened or evaluated in any method of the invention according to
any techniques known to those of skill in the art. The array format
is not required for the screening and diagnostic methods to be
implemented.
[0257] The kits for using miRNA arrays for therapeutic, prognostic,
or diagnostic applications and such uses are contemplated by the
inventors herein. The kits can include an miRNA array, as well as
information regarding a standard or normalized miRNA profile for
the miRNAs on the array. Also, in certain embodiments, control RNA
or DNA can be included in the kit. The control RNA can be miRNA
that can be used as a positive control for labeling and/or array
analysis.
[0258] The methods and kits of the current teachings have been
described broadly and generically herein. Each of the narrower
species and sub-generic groupings falling within the generic
disclosure also form part of the current teachings. This includes
the generic description of the current teachings with a proviso or
negative limitation removing any subject matter from the genus,
regardless of whether or not the excised material is specifically
recited herein.
Example XI
Array Preparation and Screening
[0259] Also provided herein are the preparation and use of miRNA
arrays, which are ordered macroarrays or microarrays of nucleic
acid molecules (probes) that are fully or nearly complementary or
identical to a plurality of miRNA molecules or precursor miRNA
molecules and that are positioned on a support material in a
spatially separated organization. Macroarrays are typically sheets
of nitrocellulose or nylon upon which probes have been spotted.
Microarrays position the nucleic acid probes more densely such that
up to 10,000 nucleic acid molecules can be fit into a region
typically 1 to 4 square centimeters.
[0260] Microarrays can be fabricated by spotting nucleic acid
molecules, e.g., genes, oligonucleotides, etc., onto substrates or
fabricating oligonucleotide sequences in situ on a substrate.
Spotted or fabricated nucleic acid molecules can be applied in a
high density matrix pattern of up to about 30 non-identical nucleic
acid molecules per square centimeter or higher, e.g. up to about
100 or even 1000 per square centimeter. Microarrays typically use
coated glass as the solid support, in contrast to the
nitrocellulose-based material of filter arrays. By having an
ordered array of miRNA-complementing nucleic acid samples, the
position of each sample can be tracked and linked to the original
sample.
[0261] A variety of different array devices in which a plurality of
distinct nucleic acid probes are stably associated with the surface
of a solid support are known to those of skill in the art. Useful
substrates for arrays include nylon, glass and silicon. The arrays
may vary in a number of different ways, including average probe
length, sequence or types of probes, nature of bond between the
probe and the array surface, e.g. covalent or non-covalent, and the
like. The labeling and screening methods described herein and the
arrays are not limited in its utility with respect to any parameter
except that the probes detect miRNA; consequently, methods and
compositions may be used with a variety of different types of miRNA
arrays.
[0262] In view of the many possible embodiments to which the
principles of our invention may be applied, it should be recognized
that the illustrated embodiments are only preferred examples of the
invention and should not be taken as a limitation on the scope of
the invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
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Sequence CWU 1
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60gauuacuugu uucuggaggc agcu 84377RNAHomo sapiens 3ccgggaccca
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ggaccgg 77422RNAHomo sapiens 4uucaaguaau ccaggauagg cu 22521RNAHomo
sapiens 5uucaaguaau ucaggauagg u 21
* * * * *
References