Biochimica et Biophysica Acta 1809 (2011) 678685

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Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g r m

Review

Role of microRNAs in hepatitis B virus replication and pathogenesis,

Wan-Hsin Liu a, b, Shiou-Hwei Yeh a, b, Pei-Jer Chen b, c, d, e,
a

Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan

NTU Center for Genomic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
c
Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
d
Hepatitis Research Center, National Taiwan University and Hospital, Taipei, Taiwan
e
Center of Genomic, Academia Sinica, Taipei, Taiwan
b

a r t i c l e

i n f o

Article history:
Received 5 March 2011
Received in revised form 24 April 2011
Accepted 25 April 2011
Available online 4 May 2011
Keywords:
MicroRNA
Hepatitis B virus
Hepatocellular carcinoma
Transcription
Replication
Pathogenesis

a b s t r a c t
The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk factor
for hepatocellular carcinoma (HCC). The role of microRNA (miRNA) in the replication and pathogenesis was
reviewed. So far none of HBV-encoded miRNA has been identied. Cellular miRNAs have shown able to
regulate HBV at the transcription level either by targeting to cellular transcriptions factors required for HBV
gene expression, or by a directly binding to HBV transcripts. We also summarized the changed patterns of
cellular miRNAs from hepatitis B progressing to cirrhosis and then liver cancer. The changing of a few of
miRNAs, such as miR-122 or miR-21, were reproduced and worthy of further research by a deep sequencing
and functional validation. These HBV-specic miRNAs should potentially become biomarkers for HBV
infection and HBV-positive HCC diagnosis. The understanding of miRNA biology paved the way for applying
miRNAs-based RNAi against HBV replication with minimal toxicities. This article is part of a Special Issue
entitled: MicroRNAs in viral gene regulation.
2011 Elsevier B.V. All rights reserved.

1. Introduction
HBV belongs to a family of small, enveloped DNA virus called
Hepadnaviridae and causes acute or persistent infection in liver. There
are more than 350 million chronic carriers of HBV worldwide and
chronic HBV infection has been strongly associated with an increased
risk of cirrhosis and in turn, leads to hepatocellular carcinoma (HCC)
[1,2]. An understanding of hepatitis B biology and pathogenesis is
important for hepatitis B control. HBV is a noncytopathic virus that
replicates preferentially in the hepatocytes. Although the mechanism
of HBV entry to hepatocytes remains unknown, the N-terminus of the
viral large envelope protein has been implicated to involve in cell
attachment and entry [3]. Following fusion of viral and cellular
membranes, the viral capsid is assumed transported to the nuclear
pore where the HBV relaxed circular genome (RC-DNA) is released
into hepatocyte nucleus. Inside the nucleus, rcDNA is converted to a
covalently closed circular DNA (cccDNA) presumably by cellular
enzymes [4,5]. The cccDNA serves as template for transcription of all
viral RNAs. HBV genome (3.2 kb) contains four overlapping open

This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.
Potential conict of interest: Nothing to declare.
Corresponding author at: Graduate Institute of Clinical Medicine, National Taiwan
University College of Medicine, No. 7 Chung-Shan South Road, Taipei 100, Taiwan. Tel.: +886
2 23123456x67072; fax: +886 2 23317624.
E-mail address: peijerchen@ntu.edu.tw (P.-J. Chen).
1874-9399/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagrm.2011.04.008

reading frames that codes for hepatitis B virus surface antigen (S),
hepatitis B virus core protein (C), viral reverse DNA polymerase (Pol)
and X protein (HBx). It also transcribes viral pregenomic RNA that will
undergo reverse transcription to synthesize viral DNA genome. The
3.5-kb transcript is slightly larger than the viral genome and encodes
for the viral core antigen and the polymerase. The 2.1-kb transcript
represents the major transcript of the S gene which encodes the viral
surface antigen and a minor singly spliced viral RNA [6]. The 2.4-kb
transcript encodes mainly the large envelope protein and a minor
doubly-spliced RNA [6]. Finally a 0.8-kb transcript encodes the HBx
protein. The viral transcripts are under the control of two master
regulators, enhancer I and II, and four distinct promoters (Fig. 1A). All
HBV mRNAs terminate at a common polyadenylation signal (Fig. 1B).
As all viral life cycles depends upon the transcription of pregenomic
and viral mRNA, so the step of HBV gene transcription has been the
key stage for understanding HBV biology.
Viruses are a class of intracellular pathogens with well established
roles in the development of many diseases. Many cellular mechanisms,
such as innate immunity and cell signaling pathways may participate in
controlling viral pathogenesis. Recently, there numerous studies
highlighted cellular or viral miRNAs as a new class of regulators in
viral pathogenesis [7]. Evidences have supported viruses, such as Herpes
viruses, encode their own miRNAs which manipulate the expression of
cellular proteins or viral proteins, facilitating their infection cycles [8].
Just as viruses-encoded miRNAs can alter the expression of host genes,
virus-host interactions have co-evolved so that viruses also could be

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679

proliferation. C/EBP can bind and activate the HBV Enhancer II in a

dose-dependent manner [15], and also binding to core promoter and S
promoter to activate their transcription [16,17]. Wang et al. showed
C/EBP- was targeted and negatively regulated by miR-155 [18],
which could down-regulate HBV transcription. A second example is
miR-372. An inhibition of miR-372 results in reducing expression
of its target gene, PRKACB, which induces phosphorylation of CAMP-response element binding protein (CREB) and dissociated CREB
from the promoter [19]. CREB was known to be required for expression
of all HBV transcription unit for its binding to viral enhancer I [20]. Finally,
a recent study by Lu's group showed miR-1 was able to enhance the HBV
core promoter transcription activity by augmenting farnesoid X receptor
alpha (FXRA) expression, a positive transcription factor [21].
HBV gene expression is found to be regulated by sex hormones and
their nuclear receptors [22]. The ligand-stimulated androgen receptor
was shown to stimulate the transcription of all HBV RNAs [23,24]. We
demonstrated the mechanism as a direct binding of androgen
receptor to the androgen-responsive element sites in viral enhancer
I [14] (Fig. 1A), a nding subsequently conrmed by other [25].
Studies from others showed that estrogen could suppress HBV gene
expression [26]. Interestingly, our group identied miR-18a overexpression in female HCC tissues, which target ER-alpha mRNA and
inhibit its protein translation [27]. An elevation of miRNA-18a might
lead to increasing HBV transcription. Moreover, we recently discovered that estrogen receptor alpha directly reduced the HBV transcription activity via interaction with enhancer I (unpublished data).
A set of miRNAs were identied to be up-regulated after testosterone
treatment in female mouse liver [28], however, none of miRNA has
been shown to regulate androgen receptor levels so far.

regulated by host-encoded miRNAs. In the case of hepatitis C, cellular

miR-122 has been shown regulating viral genome replication. In this
review, we will discuss the effect of cellular miRNAs directly or
indirectly on HBV transcription and replication, the alteration of host
miRNAs prole by HBV infection, and as well as the role of these cellular
miRNAs in the HBV-related pathogenesis.
2. Cellular miRNAs regulating HBV gene transcription and
replication
Some cellular miRNAs were found to be capable of inhibiting or
stimulating viral replication by directly targeting to viral RNAs [8,9].
Although HBV is a DNA virus, its transcripts might be targeted and
regulated by cellular miRNAs. In the following, we will focus on some
cellular miRNAs reported to affect HBV gene expression and replication.
2.1. MiRNAs modulating HBV-associated cellular proteins
HBV is complicatedly controlled within the host hepatocyte by a
number of cellular proteins at multiple steps of the HBV replication
cycle. HBV uses cccDNA to establish persistent infection by formation
of nuclear minichromosome and to serve as a transcription template
for generating pregenome and mRNA. Transcription of HBV cccDNA
was known to be tightly regulated by epigenetic mechanisms, such as
DNA methylation [10,11], acetylation, or histone modications [12]. A
number of liver-enriched transcription factors and nuclear receptors,
including STAT3, HNF1/4, also have been shown to bind HBV
promoter/enhancer elements or to be critical in regulation of HBV
transcription [13,14]. Control of HBV at the step of transcription thus
inuences both HBV gene expression and replication. Here, we will
briey discuss recent ndings about these critical transcription factors
modulated potentially by cellular miRNAs in host hepatocytes, so
affecting HBV transcription, as summarized in Fig. 1A.
CAAT enhancer-binding protein (C/EBP) and are co-expressed
in hepatocytes and are involved in the hepatocyte metabolism and

2.2. Screening for cellular miRNAs targeting to HBV transcripts

In an attempt to systemically screen for cellular miRNAs affecting
HBV replication, Zhang et al. employed a loss of function approach by
transfecting antagomirs targeting to 328 human miRNAs into HepG2

miRmiR-18a
18a

(A)
preC/C

miRmiR-372

miRmiR-155

miRmiR-155

miRmiR-1

preS1

preS2/S
C/EBP

FXRA C/EBP

ER
AR

HBV DNA

preC/Cp
C/EBP
Enh II

CREB
Enh I

ARE

DR1

miRmiR-155

DR2

DR1

(B)
miRmiR-210

3.5kb RNA
2.4kb RNA
2.1kb RNA
0.7kb RNA

miRmiR-199199-3p miRmiR-125a
125a-5p

Core/
Core/Pol
PrePre-S1
PrePre-S2/S
HBx
miR-345

miR-7
miR-196b
miR-511

miR-433
miR-205

Fig. 1. A summary of cellular miRNAs effect on HBV transcription. (A) Angled arrows indicate the HBV RNA start site for the major viral transcripts (Pre/core, PreS1, PreS, and X), and
viral enhancer I and II are schematically depicted as boxes. DR I and DR II are two short repeats essential for viral replication. ARE represents two androgen response elements were
identied to locate at enhancer I region. Transcription factors and nuclear receptors binding position on HBV genome are shown (C/EBP, CAAT enhancer-binding protein; CREB,
C-AMP-response element binding protein; AR, androgen receptor; ER, estrogen receptor; FXRA, farnesoid X receptor alpha). The miRNAs targeting cellular factors known for
regulating HBV transcription are shown. Arrow indicated an activated but the bars indicated an inhibited effect of the miRNAs on HBV transcription. (B) Positions of binding
sequences in HBV transcripts proposed to be targeted by miRNAs are shown. Italic gray color represents miRNAs were predicted to probably bind HBV transcripts by four wellestablished target-prediction software.

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2.2.15 cells which contained a dimer of the complete HBV genome

able supporting full HBV replication, and then measured HBsAg
expression [29]. In comparison to controls, antagomirs for six miRNAs
were found to cause 1.4- to 1.6-fold increase in HBsAg expression,
without signicantly affecting host cell proliferation. Bioinformatics
analysis of six miRNAs with ViTa database suggested a putative
binding site for miR-199a-3p in the HBsAg coding region and the
other binding site for miR-210 in the HBV pre-S1 region (Fig. 1B). The
direct effect of these two miRNAs on HBV RNA transcripts was
validated by GFP reporter assay. This study suggests up-regulation of
miR-199a-3p and miR-210 in HepG2 2.2.15 cells resulting in
reduction of HBV replication by a direct binding to the viral RNAs.
In addition, Russo's group found miR-125a-5p is able to interact with
HBV surface antigen and interfere with its expression, thus reducing
the amount of secreted HBsAg [30].
Consensus-target prediction approach using computer software
also can be a useful and fast tool to predict potentially critical
sequences of the HBV genome as human miRNAs target. Wu et al. used
four well-established miRNA target-prediction software, namely
miRanda, RNAhybrid, DIANA-MicroT, and MicroInspector to predict
the targets for human miRNAs in HBV sequence [31]. The analysis
showed that the miRNAs miR-7, miR-196b, miR-433 and miR-511
target the sequences in viral polymerase and surface gene; whereas
miR-205 and miR-345 target to HBX and the pre-core genes,
respectively (Fig. 1B, italic number). The target regions were highly
conserved across the various clades of HBV and could be interesting
for experimental validation. However, it should be noted the miRNAs,
if actually binding to the proposed sites, could bind to all viral RNAs,
and thus suppressing the translation of all HBV proteins and inhibiting
replication from pre-genomic RNA.
2.3. Cellular miRNA regulating HBV replication
Other than miRNAs affecting on viral translation, there are reports
on HBV replication. In Qiu's study, silence of miR-122 in HBVtransfected Huh-7 cells with antisense miR-122 signicantly increase
HBsAg and HBeAg secretion [32]. In contrast, miR-122 gain-of
function in HBV-transfected HepG2 cells which containing low
endogenous miR-122 expression showed a marked reduction of
HBsAg and HBeAg expression. Interestingly, they noticed another
important mechanism for the miR-122 effect. The miR-122 inhibitor
also caused an increase in cellular Heme oxygenase-1 (HO-1)
expression at both the mRNA and protein level [32]. HO-1 has been
show able to reduce the stability of the HBV core protein, thereby
decreasing HBV cccDNA levels both in vitro and in vivo [33]. Overall,
miR-122 was found to suppress HBV replication indirectly through
HO-1. This nding is similar with the Shan's observation on HCV
previously [34]. Finally, several lines of evidence have suggested that
miR-122 play a role in stimulating the replication and expression of
HCV, nevertheless, Qiu's study provided an opposite function of this
liver-rich miR-122 in its ability to restrict HBV replication (Fig. 2).
DNA hypermethylation might play crucial roles not only in
silencing of host genes but also in suppressing HBV cccDNA
transcription. Increased protein expression of DNMTs has been
signicantly correlated with the malignant potential [35] as well as
decrease of HBV gene expression and replication by methylating viral
DNA [10,11]. To study the relationship between HBV and DNMT
expression, Zhang's et al. showed the expression of miR-152 to be
down-regulated in the livers of HBx transgenic mice [36]. In the
further study, DNMT-1 was found to be as a target for both miR-148a
and miR-152 [37,38] (Fig. 2). MiR-152 expression was also shown to
be inversely correlated with DNMT1 expression in HBV-related HCC.
These support that miR-152 may be a factor in the regulating the
methylation of host and viral cccDNA.
Recent study by Lu's group noted that miR-1 over-expression
resulted in a marked increase of HBV replication, accompanied with

miRmiR-1

HO-1
HDAC4

HBV

DNMT1
?

miRmiR-122

miR148a
miR-148a
miRmiR-152

DR1
DR2

miRmiR-449a
449a

miRmiR-210

Fig. 2. Schematic illustration for the effects of cellular miRNA regulation on HBV
replication. The miRNAs can regulate HBV replication through modulating critical
cellular factors. MiR-1 increases viral replication by activating farnesoid X receptor
alpha (FXAR) and suppressing histone deacetylase 4 (HDAC4). MiR-122-induced
down-regulation of Heme oxygenase-1 (HO-1) negatively affects miR-122-mediated
suppression of HBV. MiR-449a enhances HBV replication, whereas miR-210 reduces its
replication by unknown cellular factors. MiR-152 and miR-148a inhibit DNA
methyltransferase 1 (DNMT1), may affecting viral replication.

up-regulated HBV transcription, antigen expression, and progeny

secretion [21]. However, miR-210 was found to decrease HBV
replication. MiR-1 was known to increase HBV transcription under
the control of the HBV core promoter in a farnesoid X receptor alpha
(FXRA)-dependent manner. Also, HDAC4, a member of histone
deacetylase, was identied as a cellular target of miR-1 and inhibited
by it. However, HDAC4 was reported to suppress HBV replication.
Thus, HDAC4 could attenuate the increase replication of HBV by miR1. Currently, they newly identied another miRNA, miR-449a, had a
high capacity for enhancing HBV replication (personal communication). But miR-449a did not target to HBV genome directly by
bioinformatic analysis and reporter assay. However, miR-449a may
promote cell differentiation by increasing expressions of hepatocytespecic factors, which may be benecial for HBV replication.
Collectively, host miRNAs can modulate HBV replication through
regulating cellular factors that do not direct bind to viral genome
(Fig. 2). This kind of indirect effect on HBV replication by cellular
miRNAs is also an important way to understand host factors involved
in HBV infection.
3. MiRNA regulated by HBV-encoded protein
Though many cellular miRNAs are reported to regulate HBV
expression and replication, however, fewer studies noticed whether
HBV-encoded proteins should inuence cellular microRNA expression.
Hepatitis B virus protein (HBx) is a 17 kDa protein encoded by HBV
and is implicated to play an important role in hepatocarcinogenesis [39].
Wang et al. studied the expression of 286 human miRNAs in HBxexpressing cells using miRNA microarrays [40]. HBx was found to
signicantly up-regulate the expression of 7 miRNAs but down-regulate
the expression of 11 cellular miRNA, respectively. An inverse correlation
was noted between the expression of HBx and that of the highlyexpressed members of the let-7 family including let-7a, let-7b and let-7c
in HCC patients. Let-7 is the rst miRNA identied in humans and was
identied to target to a number of important cellular oncogenes, such as
Ras [41], HMGA2 [42] and MYC [43]. Furthermore, STAT3, known to play
critical role in liver regeneration and HCC oncogenesis, was identied as
the direct cellular target of let-7 in Wang's study. Viral proteins other
than HBx have not been shown directly inuencing cellular miRNA
proles at present.
4. Alteration of miRNAs prole in tissues from chronic hepatitis B
patients
Recent evidences have supported that viruses might have evolved
strategies to prevent infected cells from undergoing apoptosis and

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escape the innate or adaptive immune responses in host cells through

regulating the expression of miRNAs [44]. HBV infection may
modulate the expression of host cellular miRNAs, which participated
in development of HBV-related liver diseases. Numerous studies on
miRNA proles in chronic hepatitis B patient tissues or in HBV-expressing
cells are reviewed (Fig. 3).

4.1. MiRNAs proling in HBV-associated clinical HCC

Several reports have examined the miRNA expression proles in
human HCC tissues and analyzed the miRNA alteration link to HBV
infection by miRNA-based microarray or sequencing. They also try to
nd if some specic miRNA is HBV-associated or HBV-specic. Ura et
al. measured the expression of 188 miRNAs in hepatic tumor and nontumor tissues obtained from 12 HBV-related and 14 HCV-related
patients. Depending upon the viral etiology, 6 miRNAs exhibited a
decreased expression in the HBV group and 13 miRNAs showed a
decreased expression in HCV group. This nding suggested a
difference in the miRNA expression caused by either HBV or HCV
infection [45]. Murakami et al. analyzed miRNA expression in 24 HCC
samples and 22 adjacent non-tumor tissues, 30 miRNAs were
signicantly differentially expressed in the HCC [46]. Ladeiro et al.
analyzed the expression of 250 miRNAs in a series of 46 malignant and
benign hepatocellular tumors, with respect to 4 normal liver tissues.
They further showed miR-96 signicantly over-expressed in HBVrelated HCC when compared to HCC from non-HBV causes [47]. In
Yeh's study, 17 miRNAs were found to be de-regulated in 80 HCC
patients' prole. Elevated miR-18a expression appeared to be
statistically associated with female gender, especially in HBV cases
(female to male ratio: 6.65). Their nding also suggested miR-18a
suppressing the translation of ER-alpha thus increasing the risk of HCC
development in women [27].
Wang and co-workers examined the miRNA expression pattern,
survival, and response to interferon in both men and women with
liver cancer [48]. A total of 455 patients with HCC who had undergone
radical tumor resection were included. Most patients in the study
were chronic HBV infection that in a majority of cases had liver
cirrhosis. Class-comparison analysis of paired tumors in HBV-related
HCC patients revealed that 7 miRNAs were differentially expressed
between men and women, and miR-26 is ubiquitously over-expressed
in non-cancerous liver tissues from HBV-positive HCC patients.
Analysis of gene network revealed that activation of signaling
pathways between nuclear factor B (NFB) and interleukin-6 was
generally observed in miR-26 reducing patients. Intriguingly, inter-

Elevated
miRmiR-602

Normal
Liver
Reduced

Hepatitis B

miRmiR-10b
10b
miRmiR-21(
21(2)
miRmiR-221
miRmiR-224
miRmiR-602

leukin-6 expression was inversely correlated with miR-26 expression.

Such a link is functionally relevant to liver cancer in the rodent model.
Takizawa's group using sequencing method to obtain a comprehensive prole and to characterize miRNA transcriptomes in HBV
associated HCC [49]. More than 314,000 reliable reads from HCC and
more than 268,000 from adjacent normal liver were obtained.
Expression proling using bioinformatics-based analysis and clone
count analysis showed several miRNAs were expressed aberrantly in
liver cancer, including miR-122, miR-21 and miR-34a. This technique
additionally can detect miRNA modications, for examples, adenosine-to-inosine in miR-376 family. The combination of sequencing
and bioinformatics may accelerate the discovery of novel miRNAs
involved in HBV related human liver cancer.
Recent studies reported that human serum/plasma contains a
number of stable miRNAs, which can potentially serve as a novel
noninvasive biomarker for disease diagnosis [5052]. Li et al.
examined the proles of miRNAs in serum samples from 210 controls,
135 HBV-, 48 HCV- and 120 HCC-affected individuals by Solexa
sequencing followed by validation with quantitative RT-PCR assay
[53]. It successfully identied a unique expression prole for HBVrelated serum miRNAs and HCC-related serum miRNAs, respectively.
Compared with control serum, 13 miRNAs were obtained with
differentially expressed in HBV serum (Fig. 3). This 13-miRNA-based
biomarker accurately discriminated HBV serum samples from
controls and HCV cases. Two of them, miR-375 and miR-92a, were
further identied as HBV specic. This is the rst approach to select
specic serum miRNA which can be used for detecting HBV-specic
cases.
4.2. MiRNAs proling in HBV-related cirrhosis or dysplastic nodules
Chronic hepatitis B frequently progress into liver cirrhosis that
predisposes to HCC. A growing number of studies have focused on the
expression proles of miRNAs during cirrhotic stage for their roles in
pathogenesis [5460]. Jiang's study showed that a large number of
miRNAs have increased expression in hepatitis positive and cirrhotic
tissues compared with the normal liver. Their data suggest that the
combination of both cirrhosis and hepatitis viral infection signicantly
enhances the alteration of primary miRNA expression [55]. Pineau et
al. proled miRNA expression from tissue samples of 21 normal livers,
90 adjacent cirrhotic livers, 104 HCC as well as 35 HCC cell lines and a
set of 12 miRNAs was linked to disease progression from normal liver
through cirrhosis to full-blown HCC [60]. Guo et al. showed changes in
miRNAs associated with hepatic stellate cells (HSCs), which is the key
event in liver brosis, revealed that 13 pathways were up-regulated

miRmiR-363
miRmiR-494
miRmiR-615615-3p
miRmiR-625

miRmiR-21(
21(3)
miRmiR-25
miRmiR-34a
34a
miRmiR-96
miRmiR-602
miRmiR-143

Cirrhosis
miRmiR-145
miRmiR-199b
199b

681

miRmiR-20a
20a
miRmiR-324324-3p
miRmiR-483

miRmiR-10a
10a
miR23a/b
miR-23a
miRmiR-92a
92a
miRmiR-99a
99a
miRmiR-122a
122a
miRmiR-125b
125b

miRmiR-150
miRmiR-223
miRmiR-342342-3p
miRmiR-375
miRmiR-423
letlet-7c

HCC
miRmiR-23a
23a
miRmiR-26
miRmiR-27a
27a

miR-122(
122(3)*
miRmiR-145 miRmiRmiR-152
miRmiR-199b
199b

Fig. 3. The microRNAs proles examined to be associated with the progressive stages from chronic hepatitis to cirrhosis and to HCC after HBV infection. The stages of HCC progression
are shown. Those miRNAs above represent those elevated, whereas below were miRNAs with reduced expression. The black-color group indicated changed miRNAs in HCC samples,
as compared with that of adjacent normal liver in HBV carriers. The red group represents miRNA proles in HBV serum with that in normal control serum. MiR-143 (blue) and
miR-152 were screened from tumor tissues in p21-HBx mice compared with adjacent non-cancerous hepatic tissues. MiR-602 (purple) was found elevated in tissue samples
collected from each stage compared with that from normal liver. Asterisk represents the number of times made from different observations for the indicated miRNA. MiR-122 was
consistently down-regulated in three different studies. MiR-21 was consistently up-regulated in different studies from stage of cirrhosis to HCC.

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and 22 pathways were down-regulated by miRNAs [61]. Next, we will

focus on discussing the miRNA expression prole in the HBV-related
cirrhosis.
Gao's study investigated the deregulation of miRNA in this multistep process in HBV-associated liver samples [54]. A panel of seven
miRNAs, which have been reported to be frequently de-regulated in
liver carcinogenesis [46,47,62], were selected to investigate their
expression during the early stage of HCC, associated with chronic HBV
infection. The results showed an increasing trend of miRNA deregulation from low-grade to high-grade dysplastic nodules and HCC.
Low-grade dysplastic nodules were characterized by frequent downregulation of miR-145 and miR-199b, whereas over-expression of
miR-21, miR-221 and miR-224 were frequently found in the small
HCCs. miR-122 was signicantly under-expressed in small HCCs
compared with cirrhotic/chronic hepatitis livers. This study provided
a concept that miRNA de-regulation is an early event and accumulate
along the multi-steps of HBV-associated hepatocarcinogenesis.
Hypersplenism is a clinical syndrome common in portal hypertension (PH) in liver cirrhosis patients. In general, cytopenia results
predominantly from the increased phagocytosis and destruction of
blood cells in splenic macrophages [63]. Li et al. determined whether
miRNA expression is altered in splenic macrophages associated with
hypersplenism due to portal hypertension in HBV-related cirrhosis
[64]. Screening 292 miRNAs expressed in splenic macrophages, 99
miRNAs were identied as differentially expressed between HS-PHT
and normal spleen, suggesting miRNAs may be involved in the
pathogenesis of HS-PHT. Among the miRNAs identied in this study,
miR-615-3p was signicantly up-regulated in hypersplenism. The
results in this study revealed miRNA should be a new layer of regulatory
mechanisms in the pathogenesis of hypersplenism in HBV-related
cirrhosis.
An important miRNA molecule which is signicantly up-regulated
in HBV-infected liver and HCC is miR-602 [65]. Yang et al. evaluated
miRNA expression in normal, chronic HBV hepatitis, HBV-positive
cirrhotic, HBV-positive HCC and corresponding adjacent non-tumor
livers using miRNA microarray. Fourteen miRNAs were aberrantly
expressed in HBV-related cirrhosis and HBV-related HCC, however,
miR-602 was found that started to increase signicantly from the
stage of HBV hepatitis and reached the highest in HCC compared with
normal liver. MiR-602 expression was validated to be up-regulated in
HepG2 2.2.15 and HepG2-HBx about 23 fold of that in HepG2, which
is consistent with the observation in clinical study. The cellular
RASSF1A was identied as the target of miR-602. RASSF1A protein
level increased while miR-602 inhibition in HepG2 cells, which
resulted in increased apoptosis and decreased cell proliferation rate.

4.3. Specic miRNA for HBV-related HCC in animal models

Zhang's study showed up-regulation of miR-143 expression
transcribed by NF-B in HBV-HCC promotes cancer cell invasion/
migration and tumor metastasis by repression of FNDC3B expression.
They further used miRNA array and real-time PCR to screen specic
candidate miRNA molecules in the livers of p21-HBx transgenic mice,
in which hepatocellular cancer developed 18 months after birth.
Among miRNAs analysis, miR-143 was under-expressed in the livers
of 10-month old transgenic mice and then signicantly up-regulated
after HCC developed especially when accompanied by tumor
metastasis to the lung. Additionally, miR-143 was dramatically upregulated in 25 HBV-HCC samples and reached eight-fold increase
among the HBV-HCC patients with lung metastasis. The ability of miR143 in promoting cell metastasis was conrmed in HBx-HepG2 cells
and in an athymic nude mouse model. Taken together, this study
suggested a possible pathway mediated by miR-143 that promotes
the metastasis of HCC derived from HBV infection in vitro and in vivo
[36].

4.4. MiRNAs proling in HBV-expressing cells

Cell culture is a simple system in which miRNAs change could be
easily and conveniently assayed after HBV infection or transfection.
Liu et al. compared the expression prole of cellular miRNAs of a
stable HBV-expressing cell line HepG2.2.15 and its parent cell line
HepG2 using miRNA microarray and Northern blot analysis [66].
Eighteen miRNAs were differentially expressed between the two cell
lines dened by a threshold of three-fold difference. Among them,
eleven miRNAs were found to be up-regulated and seven were downregulated in HepG2.2.15 cells. These miRNAs screened by microarray
were validated further by Northern blot analysis. The data conrmed
that the expression of miR-181a, miR-181b, miR-200b and miR-146a
were up-regulated, whereas the expression of miR-15a were downregulated. However, interactions between the HBV and the host
cellular miRNAs are largely unclear.
In summary, HBV infection was found to change the cellular miRNAs
expression in host cells (Fig. 3). During the progression from chronic
hepatitis to cirrhosis and HCC, some miRNAs were found to be
aberrantly expressed from early stage to the end HCC, such as miR-21,
miR-199b, miR-145 and miR-602. Detailed analysis also suggested that
down-regulation of miR-145 and miR-199b is an early event frequently
detected in pre-malignant dysplastic nodules and persistent throughout
HCC development [54]. Among these miRNAs, miR-145 was abundantly
expressed in non-tumorous livers but dramatically reduced in premalignant low-grade dysplastic nodules. An over-expression of miR145 in both HepG2 and Hep3B cells signicantly inhibited cell
proliferation, migration and invasion.
Many studies yielded different changing proles of miRNAs with a
greater variation, however, only both miR-122 and miR-21 changes
are consistently reproduced. MiR-122 was highly expressed in the
normal liver and cirrhosis, but signicantly down-regulated at the
stage of HCC. It is a mammalian liver-specic microRNA and is
expressed at a high level, accounting for around 70% of all cloned
miRNAs in liver [67,68]. Several studies have reported the diversity of
its roles in the hepatic function, including cell cycle progression and
tumorigenesis [69,70], lipid metabolism [71], response to stress [72],
and enhancing HCV replication [73]. The study by Tsou's group also
showed restoration of miR-122 signicantly reduced in vitro migration, invasion, and anchorage-independent growth as well as in vivo
tumorigenesis, angiogenesis, and intrahepatic metastasis by direct
suppression of the metastasis-related gene, ADAM17 [74]. Its role in
HBV-related pathogenesis deserves a more extensive study.
Two recent studies reported miR-21 up-regulation at cirrhotic
stage [54,60] and other studies showed its up-regulation was found in
HCC [49,75,76]. Obviously, miR-21 expression increased through the
progression into HCC. MiR-21's function in HCC was rstly suggested
contributing to HCC growth and spread by reducing PTEN expression
and enhancing cell growth, migration and invasion [75]. Mori's group
suggested miR-21 rendering HCC cells more resistant to IFN-alpha/5-FU
treatment and miR-21 expression in clinical HCC specimens was
associated with the clinical response to the IFN-alpha/5FU [77].
5. Application of miRNAs or antagomir for HBV therapeutic
The use of RNA interference (RNAi) to inhibit gene expression is
potentially applicable in the treatment of viral infections such as HBV
[78]. Both synthetic and expressed sequences are being widely used to
achieve RNAi-mediated HBV gene knockdown. However, a recent
nding suggested that anti-HBV shRNAs may cause serious toxicity in
vivo [79], raising an important concern of safety using expressed RNAi
sequences for therapeutic application. In order to improve the safety
of RNAi-mediated inhibition of HBV infection, other strategies were
developed to take the advantages of the use of cellular miRNAs.
In order to generate a safety and effective RNAi trigger, McCaffrey
group produced HBV RNAi triggers that more closely mimicked the

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683

Table 1
Cellular targets of miRNAs and effects on HBV biology/pathogenesis.
miRNAs

Known functions

miR-1

Inhibit cell proliferation

Cellular targets Effects on HBV biology or pathogenesis

HDAC4
E2F5
miR-10b
Promote metastasis in breast cancer
HOXD10
miR-18a
Increase HCC cell proliferation
ERalpha
miR-21
Promote proliferation and metastasis
PTEN
miR-26
Inverse correlation with NFB and IL-6
Unknown
miR-34a
Inhibit metastasis
c-Met
miR-122
Response to nutrient or toxic stress
CAT-1
Suppress hepatocarcinogenesis
Cyclin G1
Enhance HCV replication
HCV RNA
Regulation of lipid metabolism
HO-1
miR-125a-5p Inhibit cell proliferation
ERBB2
miR-152
Induces aberrant DNA methylation
DNMT-1
miR-155
Promote HCC cell growth
C/EBP
miR-199-3p Promote liver brosis
Unknown
miR-210
Accelerate progression of cholangiocarcinoma Unknown
miR-221
Regulate cell growth and apoptosis
DDIT4, Bmf
miR-223
Inhibit cell viability
STMN1
miR-224
Regulate cell proliferation and metastasis
PAK4, MMP9
miR-449a
Promote cell differentiation
Unknown
miR-602
Regulate cell proliferation and apoptosis
RASSF1A
let-7
Inhibit cell proliferation,
STAT3, MYC
Regulation of cell death
Caspase 3
Regulation of metastasis
RAS
Inhibit cell growth
HMGA2

References

Increase HBV replication by augmenting FXRA activity

[21]

Elevation in cirrhosis
Elevation in female HCC
Elevation in HCC
Down-regulation in HCC
Aberrant expression in HCC
Regulate HBV expression through inhibiting HO-1 expression

[49,54,84]
[27]
[75]
[48]
[54,85,86]
[32,6871,87]

Down-regulation in HCC
Suppress HBsAg expression
Down-regulation in HCC
Elevation in HCC
Suppress HBV replication by binding to sequences of HBsAg region
Suppress HBV replication by binding to sequences of pre-S1 region
Elevation in HCC tumorigenesis
Aberrant expression in HCC
Elevation in HCC
Enhance HBV replication by unknown mechanism
Elevation in HCC progression
Aberrant expression in HCC
Repression by HBx

[30,88]
[37,38]
[18]
[29,89]
[29,90]
[54,60,91]
[53,92]
[54,93]
Personal communication
[65]
[4043,53]

endogenous substrate of the miRNA processing pathway [78,80]. The

RNAi triggers were embedded within the contest of the endogenous
miR-30, and in total 14 miRNA-based triggers were constructed and
tested in culture. All rationally designed HBV-miRNAs triggers
reduced the levels of HBV RNA by N50% and the combination of
these strategies produced highly potent HBV RNAi. In HBV transgenic
mice model, miRNA-based HBV RNAi expression from AAV vectors
reduced about 100-fold HBV genomic DNA in serum. In notice, none of
the mice displayed any sign of overt toxicity and none died.
For another example, Arbuthnot and colleagues designed pri-miRNA
expression cassettes by replacing guide and complementary sequences of
natural miR-31 and miR-122 with those of an effective anti-HBV shRNA
[81]. The pri-miR-like sequences effectively knocked down markers of
viral replication (N80%) in cultured cells. The number of circulating viral
particle equivalents decreased at least 95% in the murine hydrodynamic
injection model. This approach used pri-miR-122 or pri-miR-31 mimic
shuttles successfully inhibit HBV replication in vitro and in vivo.
Employing a similar strategy, Jiang's group developed a lentiviral
miRNA-based system that expressed siRNAs targeting the HBsAg gene of
HBV. This construct signicantly inhibited the HBsAg mRNA and protein
level in the HepG2.2.15 cells, while HBsAg secretion was decreased by
70%. It suggests this system with lentiviral microRNA-based RNAi against
the HBsAg gene is an effective tool in controlling HBV expression [82].
Another improvement is the use of linear DNA. Since linear DNA
traverses nucleopores efciently and may therefore be useful for
improving delivery efciency [83]. Chattopadhyay et al. developed an
alternative DNA template containing anti-HBV miR-122 expression
cassettes. Silencing of HBV replication was initially assessed in cell
culture and the efciency of N75% was found without inducing an
interferon response. And a knockdown of approximately 95% HBV
replication was achieved in the hydrodynamic infection model.
Collectively, this kind of design is a useful alternative for silencing
of HBV replication in both in vitro and in vivo.
6. Conclusion and perspective
In this review, we focus on the roles of miRNAs involved in HBV
transcriptions (Fig. 1) and replication (Fig. 2). Indeed, only two miRNAs,
miR-210 and miR-199-3p, were experimentally shown to affect HBV
gene expression and replication in cell culture, maybe through a direct

binding to HBV transcripts. Their activities in HBV-infection tissues

await further studies. In contrast, there are many cellular miRNA
indirectly regulating HBV life cycles by inuencing virus-relevant
cellular proteins. Such miRNAs can also play important roles in hepatitis
B pathogenesis. We listed the cellular functions of these relevant
miRNAs in liver disease and their biological properties associated with
HBV or HBV-related HCC in Table 1. We also summarized the known
changes of miRNAs involved in multiple steps of chronic hepatitis B,
from hepatitis to cirrhosis and nally to HCC (Fig. 3). Most of these
miRNAs do not target to HBV genomes or mRNAs, but more likely to
cellular RNAs to exert their pathological effects. Obviously, certain HBVspecic host miRNAs expressions screening from serum samples were
found to be changed in the early stages during HCC development,
therefore, they should be good candidates for diagnostic biomarkers for
liver diseases prediction. Actually, most studies addressed the effect of
host cellular miRNAs on HBV infection, particularly at the level of HBV
transcription. Despite the identication of numerous cellular miRNAs
interacting with HBV, an in depth functional understanding is still
incomplete. A combination of bioinformatics and experimental screening (such as systemic antagomirs) will let us reach this goal more
effectively. The advance of the knowledge can be coupled to RNA
interference for silencing HBV infection. Ideally, the development would
apply a miRNA-based RNAi, not only achieving an effective inhibition of
HBV replication but also remaining safe in vivo. Taken together, this
understanding between miRNAs and HBV interaction will continue
grow by incorporating new miRNA and their new functions, and
possibly yielding new insights of therapeutic relevance.
Acknowledgments
This work was supported by grant from the National Research
Program of Genomic Medicine, the National Science Council, (NSC993112-B-002-015, NSC98-3112-B-002-044 and NSC99-3112-B-002-024),
the National Health Research Institutes, (NHRI-EX98-9832BI), and Center
of Genomic Medicine, Academia Sinica in Taiwan.
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