Biochimica et Biophysica Acta 1809 (2011) 678–685

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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, Taiwanb NTU Center for Genomic Medicine, National Taiwan University College of Medicine, Taipei, Taiwanc Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwand Hepatitis Research Center, National Taiwan University and Hospital, Taipei, Taiwane Center of Genomic, Academia Sinica, Taipei, Taiwan

☆ This article is part of a Special Issue entitled: Micro☆☆ Potential conflict of interest: Nothing to declare.

⁎ Corresponding author at: Graduate Institute of ClinUniversity College ofMedicine,No. 7 Chung-Shan SouthRoa2 23123456x67072; fax: +886 2 23317624.

E-mail address: peijerchen@ntu.edu.tw (P.-J. Chen).

1874-9399/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.bbagrm.2011.04.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 March 2011Received in revised form 24 April 2011Accepted 25 April 2011Available online 4 May 2011

Keywords:MicroRNAHepatitis B virusHepatocellular carcinomaTranscriptionReplicationPathogenesis

The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk factorfor hepatocellular carcinoma (HCC). The role of microRNA (miRNA) in the replication and pathogenesis wasreviewed. So far none of HBV-encoded miRNA has been identified. Cellular miRNAs have shown able toregulate HBV at the transcription level either by targeting to cellular transcriptions factors required for HBVgene expression, or by a directly binding to HBV transcripts. We also summarized the changed patterns ofcellular miRNAs from hepatitis B progressing to cirrhosis and then liver cancer. The changing of a few ofmiRNAs, such as miR-122 or miR-21, were reproduced and worthy of further research by a deep sequencingand functional validation. These HBV-specific miRNAs should potentially become biomarkers for HBVinfection and HBV-positive HCC diagnosis. The understanding of miRNA biology paved the way for applyingmiRNAs-based RNAi against HBV replication with minimal toxicities. This article is part of a Special Issueentitled: MicroRNAs in viral gene regulation.

RNAs in viral gene regulation.

ical Medicine, National Taiwand, Taipei 100, Taiwan. Tel.:+886

ll rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

HBV belongs to a family of small, enveloped DNA virus calledHepadnaviridae and causes acute or persistent infection in liver. Thereare more than 350 million chronic carriers of HBV worldwide andchronic HBV infection has been strongly associated with an increasedrisk of cirrhosis and in turn, leads to hepatocellular carcinoma (HCC)[1,2]. An understanding of hepatitis B biology and pathogenesis isimportant for hepatitis B control. HBV is a noncytopathic virus thatreplicates preferentially in the hepatocytes. Although the mechanismof HBV entry to hepatocytes remains unknown, the N-terminus of theviral large envelope protein has been implicated to involve in cellattachment and entry [3]. Following fusion of viral and cellularmembranes, the viral capsid is assumed transported to the nuclearpore where the HBV relaxed circular genome (RC-DNA) is releasedinto hepatocyte nucleus. Inside the nucleus, rcDNA is converted to acovalently closed circular DNA (cccDNA) presumably by cellularenzymes [4,5]. The cccDNA serves as template for transcription of allviral RNAs. HBV genome (3.2 kb) contains four overlapping open

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 willundergo reverse transcription to synthesize viral DNA genome. The3.5-kb transcript is slightly larger than the viral genome and encodesfor the viral core antigen and the polymerase. The 2.1-kb transcriptrepresents the major transcript of the S gene which encodes the viralsurface antigen and a minor singly spliced viral RNA [6]. The 2.4-kbtranscript encodes mainly the large envelope protein and a minordoubly-spliced RNA [6]. Finally a 0.8-kb transcript encodes the HBxprotein. The viral transcripts are under the control of two masterregulators, enhancer I and II, and four distinct promoters (Fig. 1A). AllHBV mRNAs terminate at a common polyadenylation signal (Fig. 1B).As all viral life cycles depends upon the transcription of pregenomicand viral mRNA, so the step of HBV gene transcription has been thekey stage for understanding HBV biology.

Viruses are a class of intracellular pathogens with well establishedroles in the development of many diseases. Many cellular mechanisms,such as innate immunity and cell signaling pathwaysmay participate incontrolling viral pathogenesis. Recently, there numerous studieshighlighted cellular or viral miRNAs as a new class of regulators inviral pathogenesis [7]. Evidences have supportedviruses, such asHerpesviruses, encode their own miRNAs which manipulate the expression ofcellular 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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regulated by host-encoded miRNAs. In the case of hepatitis C, cellularmiR-122 has been shown regulating viral genome replication. In thisreview, we will discuss the effect of cellular miRNAs directly orindirectly on HBV transcription and replication, the alteration of hostmiRNAs profile by HBV infection, and aswell as the role of these cellularmiRNAs in the HBV-related pathogenesis.

2. Cellular miRNAs regulating HBV gene transcription andreplication

Some cellular miRNAs were found to be capable of inhibiting orstimulating viral replication by directly targeting to viral RNAs [8,9].Although HBV is a DNA virus, its transcripts might be targeted andregulated by cellular miRNAs. In the following, we will focus on somecellularmiRNAs 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 anumber of cellular proteins at multiple steps of the HBV replicationcycle. HBV uses cccDNA to establish persistent infection by formationof nuclear minichromosome and to serve as a transcription templatefor generating pregenome and mRNA. Transcription of HBV cccDNAwas known to be tightly regulated by epigenetic mechanisms, such asDNAmethylation [10,11], acetylation, or histone modifications [12]. Anumber of liver-enriched transcription factors and nuclear receptors,including STAT3, HNF1/4, also have been shown to bind HBVpromoter/enhancer elements or to be critical in regulation of HBVtranscription [13,14]. Control of HBV at the step of transcription thusinfluences both HBV gene expression and replication. Here, we willbriefly discuss recent findings about these critical transcription factorsmodulated potentially by cellular miRNAs in host hepatocytes, soaffecting HBV transcription, as summarized in Fig. 1A.

CAAT enhancer-binding protein (C/EBP) α and β are co-expressedin hepatocytes and are involved in the hepatocyte metabolism and

(B)

(A)

HBV DNA

DR1

preS1preC/C

C/EBP

miRmiR--155155

pre

CFXRA

miRmiR--11

miR-345miR-7

miR-196bmiR-511

3.5kb RNA2.4kb RNA2.1kb RNA0.7kb RNA

CoreCore//PolPolPrePre--SS11

PrePre--SS22//SS

miRmiR--210210

Fig. 1. A summary of cellular miRNAs effect on HBV transcription. (A) Angled arrows indicateviral enhancer I and II are schematically depicted as boxes. DR I and DR II are two short repeaidentified to locate at enhancer I region. Transcription factors and nuclear receptors bindinC-AMP-response element binding protein; AR, androgen receptor; ER, estrogen receptor;regulating HBV transcription are shown. Arrow indicated an activated but the bars indicasequences in HBV transcripts proposed to be targeted by miRNAs are shown. Italic gray coestablished target-prediction software.

proliferation. C/EBP can bind and activate the HBV Enhancer II in adose-dependentmanner [15], and also binding to core promoter and Spromoter to activate their transcription [16,17]. Wang et al. showedC/EBP-β was targeted and negatively regulated by miR-155 [18],which could down-regulate HBV transcription. A second example ismiR-372. An inhibition of miR-372 results in reducing expressionof its target gene, PRKACB, which induces phosphorylation of C-AMP-response element binding protein (CREB) and dissociated CREBfrom the promoter [19]. CREBwas known to be required for expressionof all HBV transcription unit for its binding to viral enhancer I [20]. Finally,a recent study by Lu's group showedmiR-1 was able to enhance the HBVcore promoter transcription activity by augmenting farnesoid X receptoralpha (FXRA) expression, a positive transcription factor [21].

HBV gene expression is found to be regulated by sex hormones andtheir nuclear receptors [22]. The ligand-stimulated androgen receptorwas shown to stimulate the transcription of all HBV RNAs [23,24]. Wedemonstrated the mechanism as a direct binding of androgenreceptor to the androgen-responsive element sites in viral enhancerI [14] (Fig. 1A), a finding subsequently confirmed by other [25].Studies from others showed that estrogen could suppress HBV geneexpression [26]. Interestingly, our group identified miR-18a over-expression in female HCC tissues, which target ER-alpha mRNA andinhibit its protein translation [27]. An elevation of miRNA-18a mightlead to increasing HBV transcription. Moreover, we recently discov-ered that estrogen receptor alpha directly reduced the HBV tran-scription activity via interaction with enhancer I (unpublished data).A set of miRNAs were identified to be up-regulated after testosteronetreatment in female mouse liver [28], however, none of miRNA hasbeen shown to regulate androgen receptor levels so far.

2.2. Screening for cellular miRNAs targeting to HBV transcripts

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

Enh II

DR1DR2

miRmiR--372372 miRmiR--155155

S2/S

Enh IAR CREB

ER

miRmiR--1818aa

X?? preC/Cp

miRmiR--155155

/EBP C/EBP

ARE

miR-205miR-433

HBxHBx

miRmiR--199199--33pp miRmiR--125125aa--55pp

the HBV RNA start site for the major viral transcripts (Pre/core, PreS1, PreS, and X), andts essential for viral replication. ARE represents two androgen response elements wereg position on HBV genome are shown (C/EBP, CAAT enhancer-binding protein; CREB,FXRA, farnesoid X receptor alpha). The miRNAs targeting cellular factors known forted an inhibited effect of the miRNAs on HBV transcription. (B) Positions of bindinglor represents miRNAs were predicted to probably bind HBV transcripts by four well-

HBV

DR1

DR2

DNMT1

miRmiR--148148aamiRmiR--152152

miRmiR--449449aa

?

HO-1miRmiR--122122

?

miRmiR--210210

miRmiR--11 HDAC4

Fig. 2. Schematic illustration for the effects of cellular miRNA regulation on HBVreplication. The miRNAs can regulate HBV replication through modulating criticalcellular factors. MiR-1 increases viral replication by activating farnesoid X receptoralpha (FXAR) and suppressing histone deacetylase 4 (HDAC4). MiR-122-induceddown-regulation of Heme oxygenase-1 (HO-1) negatively affects miR-122-mediatedsuppression of HBV. MiR-449a enhances HBV replication, whereas miR-210 reduces itsreplication by unknown cellular factors. MiR-152 and miR-148a inhibit DNAmethyltransferase 1 (DNMT1), may affecting viral replication.

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2.2.15 cells which contained a dimer of the complete HBV genomeable supporting full HBV replication, and then measured HBsAgexpression [29]. In comparison to controls, antagomirs for six miRNAswere found to cause 1.4- to 1.6-fold increase in HBsAg expression,without significantly affecting host cell proliferation. Bioinformaticsanalysis of six miRNAs with ViTa database suggested a putativebinding site for miR-199a-3p in the HBsAg coding region and theother binding site for miR-210 in the HBV pre-S1 region (Fig. 1B). Thedirect effect of these two miRNAs on HBV RNA transcripts wasvalidated by GFP reporter assay. This study suggests up-regulation ofmiR-199a-3p and miR-210 in HepG2 2.2.15 cells resulting inreduction of HBV replication by a direct binding to the viral RNAs.In addition, Russo's group found miR-125a-5p is able to interact withHBV surface antigen and interfere with its expression, thus reducingthe amount of secreted HBsAg [30].

Consensus-target prediction approach using computer softwarealso can be a useful and fast tool to predict potentially criticalsequences of the HBV genome as humanmiRNAs target.Wu et al. usedfour well-established miRNA target-prediction software, namelymiRanda, RNAhybrid, DIANA-MicroT, and MicroInspector to predictthe targets for human miRNAs in HBV sequence [31]. The analysisshowed that the miRNAs miR-7, miR-196b, miR-433 and miR-511target the sequences in viral polymerase and surface gene; whereasmiR-205 and miR-345 target to HBX and the pre-core genes,respectively (Fig. 1B, italic number). The target regions were highlyconserved across the various clades of HBV and could be interestingfor 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 inhibitingreplication from pre-genomic RNA.

2.3. Cellular miRNA regulating HBV replication

Other than miRNAs affecting on viral translation, there are reportson HBV replication. In Qiu's study, silence of miR-122 in HBV-transfected Huh-7 cells with antisense miR-122 significantly increaseHBsAg and HBeAg secretion [32]. In contrast, miR-122 gain-offunction in HBV-transfected HepG2 cells which containing lowendogenous miR-122 expression showed a marked reduction ofHBsAg and HBeAg expression. Interestingly, they noticed anotherimportant mechanism for the miR-122 effect. The miR-122 inhibitoralso caused an increase in cellular Heme oxygenase-1 (HO-1)expression at both the mRNA and protein level [32]. HO-1 has beenshow able to reduce the stability of the HBV core protein, therebydecreasing HBV cccDNA levels both in vitro and in vivo [33]. Overall,miR-122 was found to suppress HBV replication indirectly throughHO-1. This finding is similar with the Shan's observation on HCVpreviously [34]. Finally, several lines of evidence have suggested thatmiR-122 play a role in stimulating the replication and expression ofHCV, nevertheless, Qiu's study provided an opposite function of thisliver-rich miR-122 in its ability to restrict HBV replication (Fig. 2).

DNA hypermethylation might play crucial roles not only insilencing of host genes but also in suppressing HBV cccDNAtranscription. Increased protein expression of DNMTs has beensignificantly correlated with the malignant potential [35] as well asdecrease of HBV gene expression and replication by methylating viralDNA [10,11]. To study the relationship between HBV and DNMTexpression, Zhang's et al. showed the expression of miR-152 to bedown-regulated in the livers of HBx transgenic mice [36]. In thefurther study, DNMT-1 was found to be as a target for both miR-148aand miR-152 [37,38] (Fig. 2). MiR-152 expression was also shown tobe inversely correlated with DNMT1 expression in HBV-related HCC.These support that miR-152 may be a factor in the regulating themethylation of host and viral cccDNA.

Recent study by Lu's group noted that miR-1 over-expressionresulted in a marked increase of HBV replication, accompanied with

up-regulated HBV transcription, antigen expression, and progenysecretion [21]. However, miR-210 was found to decrease HBVreplication. MiR-1 was known to increase HBV transcription underthe control of the HBV core promoter in a farnesoid X receptor alpha(FXRA)-dependent manner. Also, HDAC4, a member of histonedeacetylase, was identified as a cellular target of miR-1 and inhibitedby it. However, HDAC4 was reported to suppress HBV replication.Thus, HDAC4 could attenuate the increase replication of HBV by miR-1. Currently, they newly identified another miRNA, miR-449a, had ahigh capacity for enhancing HBV replication (personal communica-tion). But miR-449a did not target to HBV genome directly bybioinformatic analysis and reporter assay. However, miR-449a maypromote cell differentiation by increasing expressions of hepatocyte-specific factors, which may be beneficial for HBV replication.

Collectively, host miRNAs can modulate HBV replication throughregulating cellular factors that do not direct bind to viral genome(Fig. 2). This kind of indirect effect on HBV replication by cellularmiRNAs is also an important way to understand host factors involvedin HBV infection.

3. MiRNA regulated by HBV-encoded protein

Though many cellular miRNAs are reported to regulate HBVexpression and replication, however, fewer studies noticed whetherHBV-encoded proteins should influence cellular microRNA expression.Hepatitis B virus×protein (HBx) is a 17 kDa protein encoded by HBVand is implicated to play an important role inhepatocarcinogenesis [39].Wang et al. studied the expression of 286 human miRNAs in HBx-expressing cells using miRNA microarrays [40]. HBx was found tosignificantly up-regulate the expression of 7miRNAs but down-regulatethe expression of 11 cellularmiRNA, respectively. An inverse correlationwas noted between the expression of HBx and that of the highly-expressedmembersof the let-7 family including let-7a, let-7band let-7cin HCC patients. Let-7 is the first miRNA identified in humans and wasidentified to target to a number of important cellular oncogenes, such asRas [41], HMGA2 [42] andMYC [43]. Furthermore, STAT3, known toplaycritical role in liver regeneration andHCC oncogenesis, was identified asthe direct cellular target of let-7 in Wang's study. Viral proteins otherthan HBx have not been shown directly influencing cellular miRNAprofiles at present.

4. Alteration of miRNAs profile in tissues from chronic hepatitis Bpatients

Recent evidences have supported that viruses might have evolvedstrategies to prevent infected cells from undergoing apoptosis and

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escape the innate or adaptive immune responses in host cells throughregulating the expression of miRNAs [44]. HBV infection maymodulate the expression of host cellular miRNAs, which participatedin development of HBV-related liver diseases. Numerous studies onmiRNAprofiles in chronic hepatitis B patient tissues or inHBV-expressingcells are reviewed (Fig. 3).

4.1. MiRNAs profiling in HBV-associated clinical HCC

Several reports have examined the miRNA expression profiles inhuman HCC tissues and analyzed the miRNA alteration link to HBVinfection by miRNA-based microarray or sequencing. They also try tofind if some specific miRNA is HBV-associated or HBV-specific. Ura etal. measured the expression of 188miRNAs in hepatic tumor and non-tumor tissues obtained from 12 HBV-related and 14 HCV-relatedpatients. Depending upon the viral etiology, 6 miRNAs exhibited adecreased expression in the HBV group and 13 miRNAs showed adecreased expression in HCV group. This finding suggested adifference in the miRNA expression caused by either HBV or HCVinfection [45]. Murakami et al. analyzed miRNA expression in 24 HCCsamples and 22 adjacent non-tumor tissues, 30 miRNAs weresignificantly differentially expressed in the HCC [46]. Ladeiro et al.analyzed the expression of 250miRNAs in a series of 46malignant andbenign hepatocellular tumors, with respect to 4 normal liver tissues.They further showed miR-96 significantly over-expressed in HBV-related HCC when compared to HCC from non-HBV causes [47]. InYeh's study, 17 miRNAs were found to be de-regulated in 80 HCCpatients' profile. Elevated miR-18a expression appeared to bestatistically associated with female gender, especially in HBV cases(female to male ratio: 6.65). Their finding also suggested miR-18asuppressing the translation of ER-alpha thus increasing the risk of HCCdevelopment in women [27].

Wang and co-workers examined the miRNA expression pattern,survival, and response to interferon in both men and women withliver cancer [48]. A total of 455 patients with HCCwho had undergoneradical tumor resection were included. Most patients in the studywere chronic HBV infection that in a majority of cases had livercirrhosis. Class-comparison analysis of paired tumors in HBV-relatedHCC patients revealed that 7 miRNAs were differentially expressedbetweenmen andwomen, andmiR-26 is ubiquitously over-expressedin non-cancerous liver tissues from HBV-positive HCC patients.Analysis of gene network revealed that activation of signalingpathways between nuclear factor κB (NFκB) and interleukin-6 wasgenerally observed in miR-26 reducing patients. Intriguingly, inter-

miRmiR--602602

miRmiR--1010bbmiRmiR--2121((22))miRmiR--221221miRmiR--224224miRmiR--602602

miRmiR--145145miRmiR--199199bb

mmmmmmmm

mmimmmm

Elevated

Reduced

Normal Liver

Hepatitis B Cirrhos

Fig. 3. ThemicroRNAs profiles examined to be associated with the progressive stages from chare shown. Those miRNAs above represent those elevated, whereas below were miRNAs withas compared with that of adjacent normal liver in HBV carriers. The red group representsmiR-152 were screened from tumor tissues in p21-HBx mice compared with adjacent nocollected from each stage compared with that from normal liver. Asterisk represents the nuconsistently down-regulated in three different studies. MiR-21 was consistently up-regulat

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 compre-hensive profile and to characterize miRNA transcriptomes in HBVassociated HCC [49]. More than 314,000 reliable reads from HCC andmore than 268,000 from adjacent normal liver were obtained.Expression profiling using bioinformatics-based analysis and clonecount analysis showed several miRNAs were expressed aberrantly inliver cancer, including miR-122, miR-21 and miR-34a. This techniqueadditionally can detect miRNA modifications, for examples, adeno-sine-to-inosine in miR-376 family. The combination of sequencingand bioinformatics may accelerate the discovery of novel miRNAsinvolved in HBV related human liver cancer.

Recent studies reported that human serum/plasma contains anumber of stable miRNAs, which can potentially serve as a novelnoninvasive biomarker for disease diagnosis [50–52]. Li et al.examined the profiles of miRNAs in serum samples from 210 controls,135 HBV-, 48 HCV- and 120 HCC-affected individuals by Solexasequencing followed by validation with quantitative RT-PCR assay[53]. It successfully identified a unique expression profile for HBV-related serum miRNAs and HCC-related serum miRNAs, respectively.Compared with control serum, 13 miRNAs were obtained withdifferentially expressed in HBV serum (Fig. 3). This 13-miRNA-basedbiomarker accurately discriminated HBV serum samples fromcontrols and HCV cases. Two of them, miR-375 and miR-92a, werefurther identified as HBV specific. This is the first approach to selectspecific serum miRNA which can be used for detecting HBV-specificcases.

4.2. MiRNAs profiling in HBV-related cirrhosis or dysplastic nodules

Chronic hepatitis B frequently progress into liver cirrhosis thatpredisposes to HCC. A growing number of studies have focused on theexpression profiles of miRNAs during cirrhotic stage for their roles inpathogenesis [54–60]. Jiang's study showed that a large number ofmiRNAs have increased expression in hepatitis positive and cirrhotictissues compared with the normal liver. Their data suggest that thecombination of both cirrhosis and hepatitis viral infection significantlyenhances the alteration of primary miRNA expression [55]. Pineau etal. profiled miRNA expression from tissue samples of 21 normal livers,90 adjacent cirrhotic livers, 104 HCC as well as 35 HCC cell lines and aset of 12 miRNAs was linked to disease progression from normal liverthrough cirrhosis to full-blownHCC [60]. Guo et al. showed changes inmiRNAs associated with hepatic stellate cells (HSCs), which is the keyevent in liver fibrosis, revealed that 13 pathways were up-regulated

iRiR--363363iRiR--494494iRiR--615615--33ppiRiR--625625

iRR--2020aaiRiR--324324--33ppiRiR--483483

miRmiR--2121((33))miRmiR--2525miRmiR--3434aamiRmiR--9696miRmiR--602602miRmiR--143143

miRmiR--1010aamiRmiR--2323aa//bbmiRmiR--9292aamiRmiR--9999aamiRmiR--122122aamiRmiR--125125bb

miRmiR--150150miRmiR--223223miRmiR--342342--33p p miRmiR--375375miRmiR--423423letlet--77cc

miRmiR--2323aamiRmiR--2626miRmiR--2727aa

miRmiR--145145miRmiR--152152miRmiR--199199bb

is HCC

miRmiR--122122((33)*)*

ronic hepatitis to cirrhosis and to HCC after HBV infection. The stages of HCC progressionreduced expression. The black-color group indicated changed miRNAs in HCC samples,

miRNA profiles in HBV serum with that in normal control serum. MiR-143 (blue) andn-cancerous hepatic tissues. MiR-602 (purple) was found elevated in tissue samplesmber of times made from different observations for the indicated miRNA. MiR-122 wased in different studies from stage of cirrhosis to HCC.

682 W.-H. Liu et al. / Biochimica et Biophysica Acta 1809 (2011) 678–685

and 22 pathways were down-regulated by miRNAs [61]. Next, we willfocus on discussing the miRNA expression profile in the HBV-relatedcirrhosis.

Gao's study investigated the deregulation of miRNA in this multi-step process in HBV-associated liver samples [54]. A panel of sevenmiRNAs, which have been reported to be frequently de-regulated inliver carcinogenesis [46,47,62], were selected to investigate theirexpression during the early stage of HCC, associated with chronic HBVinfection. The results showed an increasing trend of miRNA de-regulation from low-grade to high-grade dysplastic nodules and HCC.Low-grade dysplastic nodules were characterized by frequent down-regulation of miR-145 and miR-199b, whereas over-expression ofmiR-21, miR-221 and miR-224 were frequently found in the smallHCCs. miR-122 was significantly under-expressed in small HCCscompared with cirrhotic/chronic hepatitis livers. This study provideda concept that miRNA de-regulation is an early event and accumulatealong the multi-steps of HBV-associated hepatocarcinogenesis.

Hypersplenism is a clinical syndrome common in portal hyper-tension (PH) in liver cirrhosis patients. In general, cytopenia resultspredominantly from the increased phagocytosis and destruction ofblood cells in splenic macrophages [63]. Li et al. determined whethermiRNA expression is altered in splenic macrophages associated withhypersplenism due to portal hypertension in HBV-related cirrhosis[64]. Screening 292 miRNAs expressed in splenic macrophages, 99miRNAs were identified as differentially expressed between HS-PHTand normal spleen, suggesting miRNAs may be involved in thepathogenesis of HS-PHT. Among the miRNAs identified in this study,miR-615-3p was significantly up-regulated in hypersplenism. Theresults in this study revealedmiRNA should be a new layer of regulatorymechanisms in the pathogenesis of hypersplenism in HBV-relatedcirrhosis.

An important miRNA molecule which is significantly up-regulatedin HBV-infected liver and HCC is miR-602 [65]. Yang et al. evaluatedmiRNA expression in normal, chronic HBV hepatitis, HBV-positivecirrhotic, HBV-positive HCC and corresponding adjacent non-tumorlivers using miRNA microarray. Fourteen miRNAs were aberrantlyexpressed in HBV-related cirrhosis and HBV-related HCC, however,miR-602 was found that started to increase significantly from thestage of HBV hepatitis and reached the highest in HCC compared withnormal liver. MiR-602 expression was validated to be up-regulated inHepG2 2.2.15 and HepG2-HBx about 2–3 fold of that in HepG2, whichis consistent with the observation in clinical study. The cellularRASSF1A was identified as the target of miR-602. RASSF1A proteinlevel increased while miR-602 inhibition in HepG2 cells, whichresulted in increased apoptosis and decreased cell proliferation rate.

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

Zhang's study showed up-regulation of miR-143 expressiontranscribed by NF-κB 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 specificcandidate 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 liversof 10-month old transgenic mice and then significantly up-regulatedafter HCC developed especially when accompanied by tumormetastasis to the lung. Additionally, miR-143 was dramatically up-regulated in 25 HBV-HCC samples and reached eight-fold increaseamong the HBV-HCC patients with lungmetastasis. The ability of miR-143 in promoting cell metastasis was confirmed in HBx-HepG2 cellsand in an athymic nude mouse model. Taken together, this studysuggested a possible pathway mediated by miR-143 that promotesthe metastasis of HCC derived from HBV infection in vitro and in vivo[36].

4.4. MiRNAs profiling in HBV-expressing cells

Cell culture is a simple system in which miRNAs change could beeasily and conveniently assayed after HBV infection or transfection.Liu et al. compared the expression profile of cellular miRNAs of astable HBV-expressing cell line HepG2.2.15 and its parent cell lineHepG2 using miRNA microarray and Northern blot analysis [66].Eighteen miRNAs were differentially expressed between the two celllines defined by a threshold of three-fold difference. Among them,eleven miRNAs were found to be up-regulated and seven were down-regulated in HepG2.2.15 cells. These miRNAs screened by microarraywere validated further by Northern blot analysis. The data confirmedthat the expression of miR-181a, miR-181b, miR-200b and miR-146awere up-regulated, whereas the expression of miR-15a were down-regulated. However, interactions between the HBV and the hostcellular miRNAs are largely unclear.

In summary, HBV infectionwas found to change the cellularmiRNAsexpression in host cells (Fig. 3). During the progression from chronichepatitis to cirrhosis and HCC, some miRNAs were found to beaberrantly expressed from early stage to the end HCC, such as miR-21,miR-199b, miR-145 and miR-602. Detailed analysis also suggested thatdown-regulation of miR-145 andmiR-199b is an early event frequentlydetected in pre-malignant dysplastic nodules and persistent throughoutHCC development [54]. Among thesemiRNAs,miR-145was abundantlyexpressed in non-tumorous livers but dramatically reduced in pre-malignant low-grade dysplastic nodules. An over-expression of miR-145 in both HepG2 and Hep3B cells significantly inhibited cellproliferation, migration and invasion.

Many studies yielded different changing profiles of miRNAs with agreater variation, however, only both miR-122 and miR-21 changesare consistently reproduced. MiR-122 was highly expressed in thenormal liver and cirrhosis, but significantly down-regulated at thestage of HCC. It is a mammalian liver-specific microRNA and isexpressed at a high level, accounting for around 70% of all clonedmiRNAs in liver [67,68]. Several studies have reported the diversity ofits roles in the hepatic function, including cell cycle progression andtumorigenesis [69,70], lipid metabolism [71], response to stress [72],and enhancing HCV replication [73]. The study by Tsou's group alsoshowed restoration of miR-122 significantly reduced in vitro migra-tion, invasion, and anchorage-independent growth as well as in vivotumorigenesis, angiogenesis, and intrahepatic metastasis by directsuppression of the metastasis-related gene, ADAM17 [74]. Its role inHBV-related pathogenesis deserves a more extensive study.

Two recent studies reported miR-21 up-regulation at cirrhoticstage [54,60] and other studies showed its up-regulation was found inHCC [49,75,76]. Obviously, miR-21 expression increased through theprogression into HCC. MiR-21's function in HCC was firstly suggestedcontributing to HCC growth and spread by reducing PTEN expressionand enhancing cell growth, migration and invasion [75]. Mori's groupsuggestedmiR-21 rendering HCC cellsmore resistant to IFN-alpha/5-FUtreatment and miR-21 expression in clinical HCC specimens wasassociated 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 ispotentially applicable in the treatment of viral infections such as HBV[78]. Both synthetic and expressed sequences are beingwidely used toachieve RNAi-mediated HBV gene knockdown. However, a recentfinding suggested that anti-HBV shRNAs may cause serious toxicity invivo [79], raising an important concern of safety using expressed RNAisequences for therapeutic application. In order to improve the safetyof RNAi-mediated inhibition of HBV infection, other strategies weredeveloped to take the advantages of the use of cellular miRNAs.

In order to generate a safety and effective RNAi trigger, McCaffreygroup produced HBV RNAi triggers that more closely mimicked the

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

miRNAs Known functions Cellular targets Effects on HBV biology or pathogenesis References

miR-1 Inhibit cell proliferation HDAC4E2F5

Increase HBV replication by augmenting FXRA activity [21]

miR-10b Promote metastasis in breast cancer HOXD10 Elevation in cirrhosis [49,54,84]miR-18a Increase HCC cell proliferation ERalpha Elevation in female HCC [27]miR-21 Promote proliferation and metastasis PTEN Elevation in HCC [75]miR-26 Inverse correlation with NFκB and IL-6 Unknown Down-regulation in HCC [48]miR-34a Inhibit metastasis c-Met Aberrant expression in HCC [54,85,86]miR-122 Response to nutrient or toxic stress CAT-1 Regulate HBV expression through inhibiting HO-1 expression [32,68–71,87]

Suppress hepatocarcinogenesis Cyclin G1Enhance HCV replication HCV RNARegulation of lipid metabolism HO-1 Down-regulation in HCC

miR-125a-5p Inhibit cell proliferation ERBB2 Suppress HBsAg expression [30,88]miR-152 Induces aberrant DNA methylation DNMT-1 Down-regulation in HCC [37,38]miR-155 Promote HCC cell growth C/EBP Elevation in HCC [18]miR-199-3p Promote liver fibrosis Unknown Suppress HBV replication by binding to sequences of HBsAg region [29,89]miR-210 Accelerate progression of cholangiocarcinoma Unknown Suppress HBV replication by binding to sequences of pre-S1 region [29,90]miR-221 Regulate cell growth and apoptosis DDIT4, Bmf Elevation in HCC tumorigenesis [54,60,91]miR-223 Inhibit cell viability STMN1 Aberrant expression in HCC [53,92]miR-224 Regulate cell proliferation and metastasis PAK4, MMP9 Elevation in HCC [54,93]miR-449a Promote cell differentiation Unknown Enhance HBV replication by unknown mechanism Personal communicationmiR-602 Regulate cell proliferation and apoptosis RASSF1A Elevation in HCC progression [65]let-7 Inhibit cell proliferation, STAT3, MYC Aberrant expression in HCC [40–43,53]

Regulation of cell death Caspase 3 Repression by HBxRegulation of metastasis RASInhibit cell growth HMGA2

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endogenous substrate of the miRNA processing pathway [78,80]. TheRNAi triggers were embedded within the contest of the endogenousmiR-30, and in total 14 miRNA-based triggers were constructed andtested in culture. All rationally designed HBV-miRNAs triggersreduced the levels of HBV RNA by N50% and the combination ofthese strategies produced highly potent HBV RNAi. In HBV transgenicmice model, miRNA-based HBV RNAi expression from AAV vectorsreduced about 100-fold HBV genomic DNA in serum. In notice, none ofthe mice displayed any sign of overt toxicity and none died.

For another example, Arbuthnot and colleagues designed pri-miRNAexpression cassettes by replacing guide and complementary sequences ofnatural miR-31 and miR-122 with those of an effective anti-HBV shRNA[81]. The pri-miR-like sequences effectively knocked down markers ofviral replication (N80%) in cultured cells. The number of circulating viralparticle equivalents decreased at least 95% in the murine hydrodynamicinjection model. This approach used pri-miR-122 or pri-miR-31 mimicshuttles successfully inhibit HBV replication in vitro and in vivo.

Employing a similar strategy, Jiang's group developed a lentiviralmiRNA-based system that expressed siRNAs targeting the HBsAg gene ofHBV. This construct significantly inhibited the HBsAg mRNA and proteinlevel in the HepG2.2.15 cells, while HBsAg secretion was decreased by70%. It suggests this systemwith lentiviralmicroRNA-based RNAi againstthe HBsAg gene is an effective tool in controlling HBV expression [82].

Another improvement is the use of linear DNA. Since linear DNAtraverses nucleopores efficiently and may therefore be useful forimproving delivery efficiency [83]. Chattopadhyay et al. developed analternative DNA template containing anti-HBV miR-122 expressioncassettes. Silencing of HBV replication was initially assessed in cellculture and the efficiency of N75% was found without inducing aninterferon response. And a knockdown of approximately 95% HBVreplication was achieved in the hydrodynamic infection model.Collectively, this kind of design is a useful alternative for silencingof 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 HBVtranscriptions (Fig. 1) and replication (Fig. 2). Indeed, only twomiRNAs,miR-210 and miR-199-3p, were experimentally shown to affect HBVgene expression and replication in cell culture, maybe through a direct

binding to HBV transcripts. Their activities in HBV-infection tissuesawait further studies. In contrast, there are many cellular miRNAindirectly regulating HBV life cycles by influencing virus-relevantcellular proteins. SuchmiRNAs can also play important roles in hepatitisB pathogenesis. We listed the cellular functions of these relevantmiRNAs in liver disease and their biological properties associated withHBV or HBV-related HCC in Table 1. We also summarized the knownchanges of miRNAs involved in multiple steps of chronic hepatitis B,from hepatitis to cirrhosis and finally to HCC (Fig. 3). Most of thesemiRNAs do not target to HBV genomes or mRNAs, but more likely tocellular RNAs to exert their pathological effects. Obviously, certain HBV-specific host miRNAs expressions screening from serum samples werefound to be changed in the early stages during HCC development,therefore, they should be good candidates for diagnostic biomarkers forliver diseases prediction. Actually, most studies addressed the effect ofhost cellular miRNAs on HBV infection, particularly at the level of HBVtranscription. Despite the identification of numerous cellular miRNAsinteracting with HBV, an in depth functional understanding is stillincomplete. A combination of bioinformatics and experimental screen-ing (such as systemic antagomirs) will let us reach this goal moreeffectively. The advance of the knowledge can be coupled to RNAinterference for silencingHBV infection. Ideally, the developmentwouldapply amiRNA-based RNAi, not only achieving an effective inhibition ofHBV replication but also remaining safe in vivo. Taken together, thisunderstanding between miRNAs and HBV interaction will continuegrow by incorporating new miRNA and their new functions, andpossibly yielding new insights of therapeutic relevance.

Acknowledgments

This work was supported by grant from the National ResearchProgram of Genomic Medicine, the National Science Council, (NSC99-3112-B-002-015, NSC98-3112-B-002-044 and NSC99-3112-B-002-024),the National Health Research Institutes, (NHRI-EX98-9832BI), and Centerof Genomic Medicine, Academia Sinica in Taiwan.

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