MicroRNA Expression in Selected Carcinomas of the Gastrointestinal Tract

Abstract

MicroRNAs (miRNAs) comprise a recently discovered class of small, 18-25 nucleotide, noncoding RNA sequences that regulate gene expression at the posttranscriptional level by binding to and inhibiting the translation of target messenger RNAs (mRNAs). Characteristic patterns of miRNA expression have been described in several malignancies of the gastrointestinal tract, and numerous investigators have demonstrated interactions between specific miRNA species and target oncogenes or tumor-suppressor genes. It is clear that miRNAs play an important role in regulating expression of a number of genes involved in gastrointestinal carcinogenesis, and, thus, these molecules may represent either diagnostic markers of, or therapeutic targets for, some types of malignancy. This paper summarizes the literature regarding miRNA expression in carcinomas of the colon, pancreas, and liver and discusses some of the mechanisms by which these molecules participate in gastrointestinal oncogenesis.

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SAGE-Hindawi Access to Research
Pathology Research International
Volume 2011, Article ID 124608, 10 pages
doi:10.4061/2011/124608
Review Article
MicroRNA Expression in Selected Carcinomas of
the Gastrointestinal Tract
Nicole C. Panarelli and Rhonda K. Yantiss
Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
Correspondence should be addressed to Rhonda K. Yantiss, rhy2001@med.cornell.edu
Received 15 September 2010; Accepted 7 January 2011
Academic Editor: Wade Samowitz
Copyright © 2011 N. C. Panarelli and R. K. Yantiss. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
MicroRNAs (miRNAs) comprise a recently discovered class of small, 18–25 nucleotide, noncoding RNA sequences that regulate
gene expression at the posttranscriptional level by binding to and inhibiting the translation of target messenger RNAs (mRNAs).
Characteristic patterns of miRNA expression have been described in several malignancies of the gastrointestinal tract, and
numerous investigators have demonstrated interactions between specific miRNA species and target oncogenes or tumor-
suppressor genes. It is clear that miRNAs play an important role in regulating expression of a number of genes involved in
gastrointestinal carcinogenesis, and, thus, these molecules may represent either diagnostic markers of, or therapeutic targets for,
some types of malignancy. This paper summarizes the literature regarding miRNA expression in carcinomas of the colon, pancreas,
and liver and discusses some of the mechanisms by which these molecules participate in gastrointestinal oncogenesis.
1. Introduction
MicroRNAs are small, 18–25 nucleotide, noncoding RNA
sequences that regulate gene expression at the posttran-
scriptional level by binding to and inhibiting translation
of target messenger RNAs (mRNAs). Over 1,000 human
miRNAs have been identified to date, and many have tissue-
specific expression profiles. Several studies have shown that
miRNAs demonstrate characteristic patterns of expression in
cancers. Some species show overexpression in cancers relative
to nonneoplastic tissues, whereas others display decreased
expression, similar to oncogenes and tumor suppressor
genes, respectively [1–9]. Emerging data indicate that virtu-
ally every type of human malignancy displays dysregulated
miRNA expression, and, in fact, potential applications of
miRNA expression profiling to the diagnosis, prognosis, and
treatment of gastrointestinal cancers have been the subject
of extensive recent investigation. Some investigators have
suggested that panels of miRNAs may be used for diagnostic
purposes among patients with suspected gastrointestinal
malignancies, and a subset of these represent prognostically
important markers or even potential therapeutic targets.
The purpose of this paper is to provide the readership with a
comprehensive overview of published data regarding miRNA
expression in gastrointestinal malignances with emphasis on
colorectal, pancreatic, and hepatocellular carcinomas.
2. Overview
MicroRNAs were first recognized as regulatory agents of gene
expression in 1993 when they were discovered in Caenorhab-
ditis elegans [10]. Each miRNA molecule can potentially bind
to either the 3-or5
-untranslated region (UTR) of hundreds
of mRNAs by sequence complementarity. Binding of miRNA
to mRNA suppresses expression by either inducing mRNA
degradation or inhibiting translational machinery. Primary
miRNA transcripts within the nucleus are processed by the
nuclear RNAse III complex, Drosha, to become miRNA
precursors termed “pre-miRNAs”. Pre-miRNAs are exported
to the cytoplasm where the endoribonuclease, Dicer, pro-
cesses them into mature miRNAs. These mature molecules
are subsequently integrated into the RNA-induced silencing
complex (RISC), the functional unit of which inhibits mRNA
translation [11,12].

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A growing body of evidence indicates that a subset of
miRNAs are functionally important to the development of
human cancers. Many investigators have identified tumor-
specific miRNA signatures that accurately distinguish malig-
nancies from benign tissues in multiple different sites, sug-
gesting that some miRNAs are oncogenic, and their potency
depends on other gene mutations that are present in the
tumor. Manipulation of miRNAs in cancer cell lines directly
affects cell proliferation and apoptosis, and many researchers
have demonstrated links between miRNA dysregulation
and cell signaling pathway abnormalities [9,13–15]. Thus,
miRNAs comprise a recently described class of molecules
that contributes to cancer formation through interactions
with mRNAs derived from oncogenes and tumor suppressor
genes.
3. MicroRNAs in Colorectal Cancer
MicroRNA expression in colon cancer and nonneoplastic
colonic tissues has been extensively studied (Tab le 1). Cum-
mins et al. performed serial analysis of gene expression
(miRAGE) in colorectal cancer cell lines and identified 133
miRAGE tags that corresponded to previously unrecognized
miRNAs. They also detected differential expression of 52
miRAGE tags in colon cancer cells relative to normal colonic
epithelium. These results provided evidence that the number
of miRNAs in the human genome is likely much larger than
had been previously predicted and that their expression is
frequently dysregulated in colorectal cancer [25]. Subsequent
studies provided data indicating that miRNA dysregulation
is important to colon cancer development. Bandres et al.
studied expression of 156 miRNAs in colon cancer cell
linesaswellaspairedtumoralandnontumoraltissues.
They identified a subset of 13 differentially expressed species
[16]. Wang et al. used miRNA microarrays to identify 12
miRNAs that were upregulated in colon cancer and 2 that
were downregulated compared to nonneoplastic colonic
tissues [17].
The expression levels of several miRNA species have been
associated with clinicopathologic features and prognosis in
colon cancer. MicroRNA-31 was first identified by Bandres
et al.as one of the most substantially dysregulated miRNAs
in colon cancer cell lines and resected colon cancers. The
authors of that study found that miR-31 expression was sig-
nificantly higher in stage IV tumors compared to stage II car-
cinomas [16]. Wang et al. later demonstrated an association
between miR-31 upregulation and advanced TNM stage as
well as deeper invasion of the primary tumor [26]. Slaby et al.
failed to identify any correlation between miR-31 expression
and tumor stage in their analysis of 29 colon carcinomas,
but they did note that miR-31 levels were significantly higher
in high-grade carcinomas, compared to low-grade tumors
[19]. MicroRNA-21, a species with antiapoptotic properties,
is dysregulated in many human cancers including tumors
of the head and neck, lung, breast, prostate, brain, thyroid,
pancreas, stomach, colon, and esophagus [9,27–32]. Its high
expression has been associated with regional lymph node
and distant metastases in colorectal cancer patients [19].
Schetter et al. analyzed 197 colonic adenocarcinomas using
microarray assays and qRT-PCR and found that high miR-
21 levels predicted poor survival prognosis and higher TNM
stage [21]. This same group later demonstrated a relationship
between high miR-21 expression and increased levels of
IL-6, a proinflammatory cytokine and lower levels of IL-
12a in colonic adenocarcinomas. They postulated that IL-
6 drives miR-21 expression whereas IL-12a is a negatively
regulated target of miR-21 [21]. Presumably, IL-12a activity
is important for host resistance to malignancy. Thus, its
downregulation by miR-21 may account for some of the
negative impact of miR-21 on prognosis among patients with
colorectal cancer. Finally, miR-145 is normally expressed in
colonic epithelium, but it shows decreased expression in
colon cancer [23]. Decreased miR-145 is more commonly
observed in tumors of the proximal colon and those of large
size (>50 mm) [22]. Akao et al. found decreased expression
of miR-143 and miR-145 in adenomas and carcinomas, but
they did not find any correlation between their expression
and any other clinical prognostic factors, indicating that
these miRNAs may primarily contribute to initiation, but
not progression, of colonic tumorigenesis. Notably, synthetic
miR-143 has a suppressive effect on growth of xenografted
tumors comprised of human colon cancer cells [33].
Expression of a number of other miRNA species has
been reported to correlate with clinicopathologic features
and prognosis among patients with colon cancer. Schepeler
et al. found that stage II colon cancers with high miR-320
or miR-498 expression showed significant differences with
respect to progression-free survival compared to tumors with
low expression of these species [18]. High expression of
miR-200c has also been reported to predict shorter survival
and is associated with frequent p53 mutations [20]. Diaz
et al. studied 110 patients with colon cancer and reported
that downregulation of miR-106a predicted shorter disease-
free survival [34]. Sarver et al. identified 6 miRNAs (HS-
29, miR-135b, miR-32, miR-33, miR-542-5p, and miR-96)
that were more highly expressed in stage IV microsatellite
stable cancers relative to stage II tumors [22]. Huang et
al. studied nonneoplastic colonic mucosa adjacent to colon
cancers in three patients with lymph node metastases and
three patients with node negative disease. They found 6-fold
higher expression of miR-137 in lymph node positive tumors
compared to node-negative cases [35]. Finally, Yantiss et al.
studied miRNA expression in colorectal cancers obtained
from 24 patients <40 years of age and 45 patients >40 years
old. They found significantly increased expression of miR-21,
miR-20a, miR-145, miR-181b, and miR-203 in tumors from
young patients compared to older adults [36].
Data from several studies have documented interactions
between specific miRNA species, oncogenes, and tumor
suppressor genes relevant to colonic carcinogenesis. Chen
et al. observed an inverse correlation between miR-143 and
KRAS expression in 13 colonic adenocarcinomas. They also
used semiquantitative RT-PCR to show that KRAS transcript
levels were decreased in cell lines transfected with pre-
miR-143 whereas addition of anti-miR-143 oligonucleotides
increased KRAS transcript levels. These findings suggest
that miR-143 downregulation in colon carcinoma promotes
cancer cell growth by disinhibiting KRAS translation [37].

Pathology Research International 3
Tab le 1: Summary of microRNA expression in colorectal cancer.
Study Differentially expressed
miRNAs (no./total)
Most significantly overexpressed miRNAs
in colorectal cancer
Most significantly underexpressed
miRNAs in colorectal cancer
Bandres et al. [16] 13/156 miR-31, miR-96, miR-133b, miR-135b,
miR-145, miR-183
Wang et al . [17] 14/723
miR-106b, miR-135b, miR-18a, miR-18b,
miR-196b, miR-19a, miR-224, miR-335,
miR-424, miR-20a∗, miR-301b, miR-734a
miR-378, miR-378∗
Schepeler et al. [18] 60/315 miR-20a, miR-510, miR-92, miR-513 miR-145, miR-455, miR-484, miR-101
Slaby et al. [19] 4/4 miR-31, miR-21 miR-145, miR-143
Xi et al. [20] 4/10 miR-15b, miR-181b, miR-191, miR-200c
Schetter et al. [21] 37/389 miR-20a, miR-21, miR-106a, miR-181b,
miR-203
Sarver et al. [22] 39/735 miR-135b, miR-96, miR-182, miR-182∗,
miR-183
miR-1, miR-133a, miR-30a-3p,
miR-30a-5p, miR-20b, miR-363
Michael et al. [23] 2/28 miR-143, miR-145
Guo et al. [24] 45/262
miR-93, miR-92, miR-520h, miR-508,
miR-505, miR-449, miR-429, miR-384,
miR-373, miR-34c, miR-326, miR-25,
miR-224, miR-210, miR-200a, miR-19b,
miR-19a, miR-18a, miR-183, miR-182,
miR-181b, miR-181a, miR-181c,
miR-17-5p, miR-148a, miR-141,
miR-130b, miR-128a, miR-106b,
miR-106a, let-7d
miR-96, miR-485-5p, miR-422b,
miR-342, miR-214, miR-199a,
miR-195, miR-150, miR-145, miR-143,
miR-133a, miR-126, miR-125b,
miR-100
MicroRNAs also impact the Wnt signaling pathway. The
adenomatous polyposis coli (APC) gene normally functions
as a tumor suppressor by regulating Wnt signaling. In
the absence of functional APC,β-catenin accumulates in
the cytoplasm and is transported to the nucleus, where it
facilitates transcription of genes involved in proliferation,
such as cyclin D1. Germline APC mutations are responsible
for familial adenomatous polyposis syndrome, and biallelic
inactivation of APC occurs in the majority of sporadic
colonic adenocarcinomas [38–41]. Transduction of colon
cancer cell lines with miR-135a and miR-135b results in
diminished APC expression and accumulation of β-catenin.
Colon cancers with high levels of miR-135a and miR-135b
show lower APC expression. In this situation, qRT-PCR
data demonstrate reduced APC mRNA, suggesting that miR-
135a and miR-135b regulate the Wnt signaling pathway by
promoting mRNA decay [42].
MicroRNA-34, a species that is lost in multiple types
of cancer including those of the colon and pancreas,
is inducible by p53, and its overexpression is associated
with p53 effects including cell cycle arrest and apoptosis
[43]. Guo et al. found that restoration of miR-126, a
species that shows low-to-absent expression in colon cancer,
impedes cancer cell growth by targeting the p85B subunit of
phosphatidylinositol-3-kinase (PI3K). Phosphatidylinositol-
3-kinase activates AKT, a protein kinase involved in the
PI3K/AKT/mTOR pathway, which triggers a variety of
downstream responses related to cell growth, proliferation,
and motility. Presumably, loss of miR-126 removes a critical
checkpoint in the PI3K/AKT/mTOR pathway and facilitates
tumor growth [24]. Continued research will likely uncover
additional regulatory roles for miRNAs in colorectal carcino-
genesis and other neoplasms throughout the gastrointestinal
tract.
Most colorectal cancers are characterized by aneuploidy,
allelic imbalance, and mutations in KRAS,TP53,andAPC
although approximately 15% of sporadic colonic adeno-
carcinomas develop via microsatellite instability (MSI) and
have defective DNA mismatch repair mechanisms. Such
tumors are often located in the proximal colon, show high-
grade histology, and contain infiltrating lymphocytes. They
are generally diploid and have a better prognosis than
non-MSI tumors, but they are probably less responsive to
conventional 5-flourouracil-based chemotherapy [44,45].
Patients with germline mutations in one of the mismatch
repair genes (MLH1,MSH2,MSH6,andPMS2)areat
risk for acquired mutations of the second allele and devel-
opment of heritable microsatellite unstable carcinomas of
the gastrointestinal, genitourinary, and gynecologic tracts,
termed Lynch syndrome. Sporadic colonic carcinomas with
MSI may also occur, but in this situation, tumorigenesis
results from biallelic epigenetic methylation and silencing of
MLH1. Several authors have studied differences in miRNA
expression patterns between microsatellite stable (MSS)
colon cancers and those with MSI-H (Tabl e 2). Lanza et al.
used microarray data to profile 23 MSS and 16 sporadic
MSI-H colon cancers. They identified 72 mRNAs and 14
miRNAs that were differentially expressed between these
two tumor types and used their combined expression to
distinguish between MSS and MSI-H cancers [14]. Sarver
et al. studied expression of 735 miRNAs in 12 MSI-H and
68 MSS colon tumors and found that miR-552, miR-592,

4Pathology Research International
Tab le 2: MicroRNA expression in microsatellite stable versus microsatellite unstable colorectal cancer.
Study MSS colorectal cancers MSI-H colorectal cancers
Overexpressed Underexpressed Overexpressed Underexpressed
Schepeler et al. [18]miR-20a, miR-510, miR-92,
miR-513
miR-142-3p, miR-212,
miR-146b, miR-217
miR-492, miR-20a,
miR-432∗, miR-21
miR-145, miR-455,
miR-484, miR-101
Sarver et al. [22]miR-552, miR-592,
miR-181c, miR-196b miR-625, miR-31
Lanza et al. [14]
miR-215, miR-192, miR-191,
miR-203, miR-32, miR-17,
miR-25, miR-106a, miR-92-1,
miR-92-2, miR-93-1, miR-20
miR-223, miR-155
miR-181c, and miR-196b were significantly increased in MSS
tumors relative to MSI-H tumors, whereas miR-625 and
miR-31 levels were higher in MSI-H tumors [22]. Schepeler
et al. compared miRNA expression in 37 MSS relative to 12
MSI-H cancers and identified a 4-miRNA signature (miR-
142-3p, miR-212, miR-151, and miR-144) that predicted
MSI with 81% specificity and 92% sensitivity [18]. These
data indicate that the type of genetic instability in colorectal
cancer is reflected at the miRNA level.
5-Flourouracil (5-FU) has been a mainstay of colorectal
cancer therapy for the past several decades [46]. Some
patients have a suboptimal response to therapy for unclear
reasons, so identification of molecular markers that predict
the likelihood of a therapeutic response is important. In
vitro studies evaluating miRNA expression in colon cancer
cell lines treated with 5-FU have generated promising
data regarding the potential use of miRNAs as markers
of chemosensitivity. Borralho et al. showed that stable
expression of miR-143, a species known to be downregulated
in colon cancer, was associated with increased death in cell
lines after exposure to 5-FU [23,47]. Others have applied
this concept to colon cancer resection specimens. Nakajima
et al. used qRT-PCR to study miRNA expression in residual
or recurrent colon cancers from 46 patients who were treated
with oral 5-FU alone or in combination with cisplatin.
Twenty-seven patients who experienced complete disease
remission showed a partial response or maintained stable
disease after treatment had significantly lower levels of let-
7g and miR-181b, compared to 19 patients who suffered
disease progression [48]. Schetter et al. analyzed associations
between miR-21 expression and therapeutic outcomes in
56 stage II or stage III colorectal cancer patients treated
with 5-FU. High miR-21 expression was associated with
worse overall survival, lending preliminary support to the
notion that miR-21 overexpression predicts a poor response
to therapy [21]. Boni et al. investigated associations between
polymorphisms in miRNA-containing genomic regions and
genes related to miRNA biogenesis and clinical outcome in
patients with metastatic colon cancer who were treated with
5-FU and irinotecan. Single-nucleotide polymorphisms in
the miR-26-a-1 gene and 5UTR of pre-miR-100 correlated
with better overall survival and disease control, respectively,
and both were associated with a prolonged interval to pro-
gression [49]. The mechanisms by which miRNAs modulate
efficacy of therapy are not understood, but these early data
support the hypothesis that changes in miRNA expression
levels and in the miRNA genome impact tumor response to
therapy.
4. MicroRNAs in Pancreatic Neoplasia
Aberrant miRNA expression has been described in pancre-
atic ductal adenocarcinoma and benign pancreatobiliary dis-
ease (Tabl e 3). Early studies exploited differences in miRNA
expression patterns to distinguish between benign and
malignant pancreatic diseases. Bloomston et al. examined
65 pancreatic ductal adenocarcinomas and benign adjacent
pancreatic tissue, as well as 42 cases of chronic pancreatitis
for miRNA expression. They found 21 miRNAs to be dysreg-
ulated in cancer compared to benign tissues and noted that a
panel of 11 miRNAs (miR-148a, miR-148b, miR-155, miR-
181a, miR-181b, miR-181b-1, miR-181c, miR-181d, miR-
21, miR-221, and miR-375) distinguished pancreatic ductal
adenocarcinoma from chronic pancreatitis and normal pan-
creatic tissue [49]. Lee et al. analyzed 28 pancreatic ductal
adenocarcinomas and 21 nonneoplastic pancreatic tissues
and found miR-155, miR-181b, miR-181c, miR-21, and miR-
221 to be among the top 20 of 100 miRNAs overexpressed
in pancreatic cancer [31]. Szafranska et al. identified 26
dysregulated miRNAs in pancreatic adenocarcinoma using
resection specimens and pancreatic cancer cell lines. They
reported that miR-196a upregulation combined with miR-
217 downregulation reliably distinguished pancreatic cancer
from benign pancreas and chronic pancreatitis [50]. In a later
study, this signature correctly identified malignancy in 9/10
fine needle aspiration biopsies of pancreatic cancer [51].
The roles of dysregulated miRNAs in neoplastic pancre-
atic lesions have also been examined. du Rieu et al. used qRT-
PCR to study miRNA expression in pancreatic intraepithelial
neoplasia (PanIN) samples from human and mouse pancreas
and found that levels of miR-21, miR-221, miR-22, and let-
7a increased in the progression of PanIN to carcinoma [52].
Dilhoffet al. found that strong miR-21 expression by in situ
hybridization was associated with decreased survival among
patients with lymph node-negative pancreatic carcinoma
[53]. Ikenaga et al. used qRT-PCR to evaluate miR-203
levels in resection specimens from patients with pancreatic
ductal adenocarcinoma (n=113), chronic pancreatitis
(n=20), and samples of nondiseased pancreas (n=8) and
found higher expression of miR-203 in pancreatic cancer.

Pathology Research International 5
Tab le 3: Summary of microRNA expression in pancreatic ductal adenocarcinoma.
Study Differentially expressed
miRNAs (no./total)
Most significantly overexpressed
miRNAs compared with normal
controls and/or chronic pancreatitis
Most significantly underexpressed
miRNAs compared with normal
controls and/or chronic pancreatitis
Bloomston et al. [30] 46/326
miR-221, miR-181a, miR-155,
miR-210, miR-213, miR-181b,
miR-222, miR-181b-2, miR-21,
miR-181b-1, miR-181c, miR-220,
miR-181d, miR-223, miR-100-1/2,
miR-125a, miR-143, miR-10a,
miR-146, miR-99, miR-100,
miR-199a-1, miR-10b, miR-199a-2,
miR-107, miR-103-2, miR-125b-1,
miR-205, miR-23b, miR-23a, miR-96,
miR-34, miR-497, miR-203, miR-453,
miR-92, miR-93, miR-21
miR-148a, miR-148b, miR-375,
miR-494, miR-483, miR-339,
miR-218-2, miR-409-3p
Lee et al. [31] 100/222
miR-221, miR-424, miR-301, miR-100,
miR-376a, miR-125b-1, miR-21,
miR-16-1, miR-181a, miR-181c,
miR-92-1, miR-15b, miR-155, let-7f-1,
miR-212, miR-107, miR-24-1,
miR-24-2, let-7d
miR-345, miR-142-p, miR-139
Szafranska et al. [50] 26/377
miR-205, miR-143, miR-145,
miR-146a, miR-148a, miR-196b,
miR-93, miR-31, miR-210, miR-196a,
miR-18a, miR-203, miR-150, miR-155,
miR-221, miR-222, miR-223, miR-224
miR-29c, miR-216, miR-217, miR-375,
miR-148a, miR-96, miR-148b,
miR-141, miR-130b
du Rieu et al. [52]7/7
miR-21, miR-221, miR-222, miR-200,
miR-205, miR-29c let-7a
Results of a multivariate analysis showed miR-203 expression
to be an independent predictor of poor prognosis in patients
who had undergone complete tumor resection [54]. Wang
et al. investigated expression of miR-21, -210, -155, and
196a by qRT-PCR in the sera of 49 patients with pancreatic
adenocarcinoma and 36 healthy controls and found higher
expression of all four markers in sera of pancreatic cancer
patients [55]. In a later study, Kong et al. analyzed serum
levels of miR-196a, miR-21, and miR-155 in 35 patients
with pancreatic adenocarcinoma, 15 patients with chronic
pancreatitis, and 15 healthy controls. They found that higher
miR-21 expression distinguished cancer patients from those
with chronic pancreatitis and healthy subjects, whereas miR-
155 and miR-196a discriminated between patients with
chronic pancreatitis and healthy controls. They also noted
that serum miR-196a levels were significantly higher in
patients with unresectable cancer than in those amenable to
surgery and that higher miR-196a levels predicted shorter
survival in pancreatic cancer patients [56]. Others have
shown increased miR-196a expression to be associated with
a 2-year survival of 17%, compared to 64% among tumors
with low expression of this marker [30].
The relative tissue-specificity of miRNAs makes them
attractive targets for molecular therapy among patients with
pancreatic cancer. Park et al. analyzed the effects of miR-21
and miR-221 antisense oligonucleotides on pancreatic cancer
cell lines and found that treated cells showed increased
apoptosis and cell cycle arrest compared to cells treated
with control oligonucleotides. MicroRNA-21 targets two
tumorsuppressormolecules,PTENandRECK,bothof
which were found to be increased in extracts from cell
lines treated with antisense oligonucleotides to miR-21.
Similarly, p27, the target of miR-221, increased when this
molecule was inhibited. Park et al. also found that cells
pretreated with antisense sequences against miR-21 and
miR-221 showed decreased viability by colorimetric analysis
following gemcitabine treatment compared to those treated
with control oligonucleotides [57].
Few studies have investigated miRNA expression in
pancreatic endocrine and acinar tumors. Roldo et al.
performed microarray and northern blot analyses of 40
endocrine tumors, 4 acinar cell carcinomas, and 12 samples
of nonneoplastic pancreas. They found that stable expression
of miR-103 and miR-107, in combination with a lack of
miR-155 expression, discriminated all tumor samples from
nonneoplastic tissues. They also identified 10 miRNAs that
distinguished endocrine from acinar tumors and 28 miRNA
species that were aberrantly increased in both tumor types
[29]. These preliminary data suggest that altered miRNA
expression occurs in endocrine and acinar neoplasms of the
pancreas.
5. MicroRNAs in Liver Disease
Early studies evaluating miRNA expression in hepatocellular
carcinoma identified approximately 69 dysregulated species

6Pathology Research International
Tab le 4: Summary of microRNA expression in hepatocellular carcinoma.
Study Differentially expressed
miRNAs (no./total)
Most significantly overexpressed
miRNAs compared with nonneoplastic
liver (no disease or chronic viral
hepatitis)
Most significantly underexpressed
miRNAs compared with nonneoplastic
liver (no disease or chronic viral
hepatitis)
Murakami et al. [58] 7/180 miR-18, miR-224 miR-199a∗, miR-195, miR-199,
miR-200a, miR-125a
Li et al. [59] 84/509 miR-106b, miR-15b, miR-18a,
miR-221, miR-222, miR-224 miR-125b, miR-101
Ladeiro et al. [67] 130/250 miR-224, miR-200c, miR-203, miR-21,
miR-222, miR-10b miR-422b, miR-122a
Varnholt et al. [60] 29/80 miR-122, miR-100, miR-10a miR-198, miR-145
Li et al. [75]8/8
miR-17-5p, miR-18a, miR-19a,
miR-20a, miR-92-1, miR-106b,
miR-93, miR-25
Pineau et al. [61] 12/215
miR-106b, miR-21, miR-210, miR-210,
miR-221, miR-222, miR-224, miR-34a,
miR-425, miR-519a, miR-93, miR-96
let-7c
(Tabl e 4)[58–63]. Many of those miRNAs, including miR-
122, miR-221, and miR-222, were later recognized as car-
cinogenic catalysts and prognostic markers in hepatocellular
carcinoma. MicroRNA-122 is the most abundant miRNA in
hepatic parenchyma and is relatively specific for hepatocyte
differentiation, showing rare expression outside of liver [64–
66]. Downregulation of miR-122 is frequently observed in
hepatocellular carcinoma, and hepatocellular carcinoma cell
lines treated with miR-122 oligonucleotides show increased
apoptosis and decreased viability [58,59,67–72]. Coulouarn
et al. correlated miR-122 tissue levels with the clinicopatho-
logic features of 64 hepatocellular carcinomas and found
that low expression of this marker predicted shorter survival,
high-grade histology, and large tumor size. They also noted
that loss of miR-122 was associated with higher expression
of genes involved in cell motility, angiogenesis, hypoxia, and
epithelial-mesenchymal transition [73]. Tsai et al. reported
lower levels of miR-122 in hepatocellular carcinomas with
intrahepatic metastases compared to solitary tumors [74].
The roles of microRNA species in hepatocellular car-
cinogenesis and their molecular targets are under cur-
rent investigation. Wong et al. found that high miR-222
levels correlated with advanced tumor stage and shorter
overall survival in patients with hepatocellular carcinoma
independent of stage. They also noted PI3K/AKT/mTOR
pathway inhibition occurred in hepatocellular carcinoma cell
lines transfected with anti-miR-222 [62]. MicroRNA-221,
another frequently dysregulated species in hepatocellular
carcinoma, participates in the modulation of key molecules
related to hepatocarcinogenesis. Pineau et al. found that
expression levels of the cyclin-dependent kinase inhibitor,
p27, and the PI3K/AKT/mTOR pathway regulator, DDIT4,
were decreased in liver cancer cell lines that overexpressed
miR-221 [61]. Gramantieri et al. showed an inverse cor-
relation between miR-221 upregulation and levels of the
proapoptotic protein, Bmf, in hepatocellular carcinoma
samples [76]. Fornari et al. showed increased CDKN1C/p57
and CDKN1B/p27 protein levels by western blot analy-
sis in hepatocellular carcinoma cell lines transfected with
anti-miR-221 compared to controls. Conversely, cell lines
treated with miR-221 showed decreased CDKN1C/p57 and
CDKN1B/p27 protein levels [77]. Finally, Mneg et al.
reported that inhibition of miR-21 in hepatocellular carci-
noma cell lines increased expression of PTEN and decreased
tumor cell proliferation, suggesting that increased miR-21
levels promote carcinogenesis [78]. Other identified, but
less-well-studied, modulators of apoptosis in hepatocellular
carcinoma include miR-29, miR-15b, miR-152, miR-101, and
the miR-106b-25 cluster [75,79–82].
Ji et al. evaluated a cohort of 214 patients with hepatocel-
lular carcinoma and found that tumors with reduced miR-
26 expression had a favorable response to adjuvant therapy
with interferon alpha, whereas those with high miR-26 did
not respond to therapy, suggesting that miR-26 may be used
to select patients who may benefit from interferon alpha
treatment [83]. Connelly et al. reported that miR-21 and
the miR-17-92 polycistron are consistently upregulated in
human and animal hepatocellular carcinoma cell lines and
that their inhibition by antisense oligonucleotides causes
reduced tumor cell proliferation [84].
Recent studies have established a role for miRNAs in
regulation of hepatitis C virus (HCV) infection and offer
promise for new treatment modalitites. The most frequently
implicated miRNA in HCV modulation is the liver-specific
species, miR-122. Jopling et al. first described a physical
interaction between miR-122 and the HCV genome by
showing that miR-122 binds to the 5UTR of viral RNA
and stimulates viral replication [85]. Henke et al. showed
that miR-122 drives HCV translation by enhancing the
association between a small ribosomal subunit and HCV
RNA [86]. Both mechanisms of HCV potentiation were later
validated by Jangra et al. who demonstrated that viruses with
mutations in miR-122 binding sites failed to replicate [87].
Young et al. reported decreased viral replication in liver cells

Pathology Research International 7
treated with inhibitors of miR-122 and suggested that these
small molecules may represent a new target for HCV therapy,
which has already been successfully tested in HCV-infected
chimpanzees [72,88].
The potential impact of miRNA analysis on patient
selection for specific therapies was underscored by Sarasin-
Filipowicz et al. These authors assessed miR-122 levels by
qRT-PCR in pre- and posttreatment liver biopsies from 42
patients with HCV. They found that patients with decreased
miR-122 levels in pretreatment liver biopsies showed a poor
response to interferon therapy [89]. Other miRNA species
such as miR-24, miR-149, miR-638, and miR-1181 have
also been implicated in HCV-related liver disease and may
facilitate viral entry, replication, and propagation [90].
MicroRNA dysregulation also occurs in association with
hepatitis B virus (HBV) infection and may provide clues to
the pathogenesis of HBV-related disease in infected patients.
Yang et al. found that miR-602 expression increased with
progression of HBV-related hepatitis to cirrhosis and hepato-
cellular carcinoma and noted that the tumor suppressor gene
RASSF1A was inhibited in cell lines that highly expressed
miR-602 [91]. Ura et al. studied 12 patients with HBV-
related hepatocellular carcinoma and 14 with HCV-related
hepatocellular carcinoma. They identified 19 differentially
expressed miRNAs between patients with HBV and HCV
infection. Microarray analysis also identified separate target
genes for HBV- and HCV-related cancers. MicroRNAs
important to HBV-related carcinoma regulate genes involved
in cell death, DNA damage, recombination, and signal trans-
duction whereas those important to HCV-related carcinoma
were related to immune response, antigen presentation, cell
cycle, and proteasome and lipid metabolism [92]. These
findings provide insight into the differences between HBV-
and HCV-infection and disease progression and may help
identify potential therapeutic target molecules in the future.
6. MicroRNAs and
In Vitro
Cancer Models
Several strategies utilizing miRNAs as in vivo therapeutic
targets are currently under development. Use of antisense
oligonucleotides has been most extensively studied in vitro,
and was recently shown to be an effective suppressor of
miRNA expression in vivo. Krutzfeldt et al. engineered
synthetic RNA analogues, termed “antagomiRs,” to miR-
16, miR-122, miR-192, and miR-194. These compounds
were administered to mice intravenously and corresponding
miRNA levels were measured by northern blot assay 24
hours after injection. These authors reported a marked
reduction in target miRNA levels in various tissues including
liver, lung, kidney, heart, intestine, fat, skin, bone marrow,
muscle, ovaries, and adrenal glands [93]. Locked nucleic acid
(LNA) constructs represent another promising approach to
suppressing miRNA expression. These molecules are nucleic
acid analogues that are “locked” by a methylene bridge
connecting the 2’O and 4’C atoms. This structural modifi-
cation enables LNA oligonucleotides to bind complementary
nucleotide sequences with high affinity and excellent mis-
match discrimination. Elmen et al. administered an LNA-
antimiRtoAfricangreenmonkeysinordertostudyits
effect on plasma cholesterol levels and miR-122 levels in
liver tissue by northern blot analysis. They observed a dose-
dependent decrease in total plasma cholesterol and depletion
of mature miR-122 in liver biopsies from these monkeys [94].
Finally, some investigators have employed adenovirus vectors
to increase expression of tumor-suppressor miRNAs. Kota
et al. showed that the adenovirus vector-mediated introduc-
tion of miR-26, a species downregulated in hepatocellular
carcinoma, into mice with liver cancer caused cancer cell
apotosis and tumor regression. This therapy had no adverse
effect on benign hepatocytes, underscoring the potential
applications of this approach [95]. Future in vivo progress
in this field will depend upon increased understanding of
miRNA function in mammals, improved chemical design of
antimiRs and synthetic miRNAs, and development of more
efficient methods for delivery of these molecules to target
tissues.
7. Conclusion
MicroRNAs represent an important class of molecules with
profound diagnostic and therapeutic implications. Emerging
evidence suggests that they may be useful diagnostic adjuncts
that aid identification of tumors of unknown origin or
even ascertain the presence of malignancy in scant biopsy
specimens or sera of patients with suspected cancer. Specific
miRNA expression profiles clearly correlate with prognosis,
so it is highly likely that miRNA analysis will play an
important role in determining the management of patients
in the future. Preliminary studies utilizing antisense oligonu-
cleotides against cancer-specific miRNAs have shown that
some tumors respond to therapy while minimally damaging
healthy tissues. These findings suggest that targeted therapies
against selected miRNAs represent a new treatment modality
for patients with gastrointestinal malignancies. Advances
in this field have improved our understanding of the
heterogeneity of human malignancies and will contribute
to the growing trend toward individualized management
strategies for cancer patients.
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