Hepatitis B Virus mRNA-Mediated miR-122 Inhibition UpregulatesPTTG1-Binding Protein, Which Promotes Hepatocellular CarcinomaTumor Growth and Cell Invasion

Changfei Li,a Yanzhong Wang,a Saifeng Wang,a Bo Wu,a Junli Hao,a Hongxia Fan,a Ying Ju,a Yuping Ding,b Lizhao Chen,a

Xiaoyu Chu,a Wenjun Liu,a Xin Ye,a Songdong Menga

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, Chinaa; Yantai City Hospital forInfectious Diseases, Yantai, Shandong, Chinab

As the most abundant liver-specific microRNA, miR-122 is involved in diverse aspects of hepatic function and neoplastic trans-formation. Our previous study showed that miR-122 levels are significantly decreased in hepatitis B virus (HBV)-infected pa-tients, which may facilitate viral replication and persistence (S. Wang, L. Qiu, X. Yan, W. Jin, Y. Wang, L. Chen, E. Wu, X. Ye,G. F. Gao, F. Wang, Y. Chen, Z. Duan, and S. Meng, Hepatology 55:730 –741, 2012). Loss of miR-122 expression in patients withhepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity.). In this study, we provide evidencethat all HBV mRNAs harboring an miR-122 complementary site act as sponges to bind and sequester endogenous miR-122, indi-cating that the highly redundant HBV transcripts are involved in HBV-mediated miR-122 suppression. We next identified pitu-itary tumor-transforming gene 1 (PTTG1) binding factor (PBF) as a target of miR-122 and demonstrated that HBV replicationcauses an obvious increase in PBF levels. Furthermore, we observed that the miR-122 levels were decreased and PBF was upregu-lated in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). Overexpression and knockdown studies both revealedthat PBF enhances proliferation and invasion of HCC cells, and silencing PBF resulted in a dramatic reduction of HCC tumorgrowth in vivo. Mechanistic analysis demonstrated that PBF interacts with PTTG1 and facilitates PTTG1 nuclear translocation,subsequently increasing its transcriptional activities. Therefore, we identified a novel HBV mRNA-miR-122-PBF regulatorypathway that facilitates malignant hepatocyte growth and invasion in CHB which may contribute to CHB-induced HCC devel-opment and progression. Our work underscores the reciprocal interplay of host miRNA sequestration and depletion by viralmRNAs, which may contribute to chronic-infection-related cancer.

MicroRNAs (miRNAs) are a large family of small (�21-nucle-otide [nt]) noncoding RNAs that interact with complemen-

tary target sites in their target mRNAs to induce translational re-pression, deadenylation, and degradation (1). However, thereciprocal effect of target mRNA on miRNA activity is largelyunknown. MicroRNA-122 (miR-122) is the most abundant liver-specific miRNA, accounting for approximately 70% of the totalmiRNA population in the adult liver (2). It has been found to playkey roles in liver development and hepatic function (3, 4), hepa-tocyte growth, neoplastic transformation and tumorigenicity (5–8), lipid metabolism (9, 10), and regulation of hepatitis B virus(HBV) and hepatitis C virus (HCV) replication (11–13).

HBV is a small (�3.2 kb), enveloped, partially double-stranded DNA virus. The HBV genome contains four overlappingopen reading frames (ORFs). The RNA transcripts are polyade-nylated; capped; 3.5, 2.4, 2.1, and 0.7 kb in length; and named thepre-C/C or pregenomic RNA (pgRNA), pre-S, S, and X mRNAs,respectively. These mRNAs encode several overlapping viral pro-teins, including the polymerase, core, HBe, pre-S1, S2, S, and Xproteins (14). There are approximately 350 million chronic HBVcarriers worldwide, and chronic HBV infection is the major etio-logical factor in hepatocellular carcinoma (HCC) (15, 16). Therelative risk for the development of HCC in chronic hepatitis B(CHB) patients is estimated to be 25 to 100 times higher than thatin those without infection (15, 17, 18).

Several possible pathways and molecular mechanisms havebeen reported for the involvement of HBV infection in malignanttransformation of liver cells, including both direct and indirect

mechanisms that likely act synergistically. Direct effects by viralfactors include HBV DNA integration into the hepatocyte genome(which acts via cis- or trans-activation of nearby genes or enhanceshost chromosomal instability), the antiapoptotic and procarcino-genic functions of the HBx and truncated pre-S2/S viral proteins,and HBV mutants and genotypes (14, 15, 19, 20). The indirecteffects of chronic viral infection on malignant transformation in-clude persistent inflammation and liver cirrhosis (which may sig-nificantly contribute to the transformation of hepatocytes andpromote hepatocarcinogenesis through an integrated multistepprocess [21, 22]), aberrant DNA methylation of specific cellulargenes (23), and host susceptibility (24). However, the molecularmechanisms underlying HBV-induced carcinogenesis remainelusive and await further investigation (14, 15).

Our previous study showed that loss of miR-122 induced byHBV infection enhances HBV replication through cyclin G1-modulated p53 activity, thereby possibly contributing to viral per-sistence (25). Moreover, miR-122 repression is only found inHCC arising in HBV-infected livers but not in HCV-infected liv-

Received 10 October 2012 Accepted 29 November 2012

Published ahead of print 5 December 2012

Address correspondence to Songdong Meng, mengsd@im.ac.cn.

C.L. and Y.W. contributed equally to this article.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.02831-12

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ers (26). Based on these observations, we aimed to explore howHBV infection leads to decreased miR-122 and whether miR-122repression contributes to the development of HCC in CHB pa-tients. In this study, we provide experimental evidence that all fourHBV mRNAs harbor an miR-122 complementary site that mayact as a sponge to bind and sequester endogenous miR-122. More-over, we identified pituitary tumor transforming gene (PTTG)binding factor (PBF; PTTG1IP) as a target of miR-122 and showedthat PBF is involved in the regulation of liver cancer cell prolifer-ation, invasion, and tumor growth. Therefore, our results reveal anovel mechanism by which viral mRNAs mediate host miRNAactivity and which contributes to chronic infection-inducedcancer.

MATERIALS AND METHODSPatients and human specimens. Liver specimens from 23 patients withCHB and 19 HBV-infected HCC liver tissues from patients undergoingHCC resection were collected for miR-122 analysis and detection of HBVmRNAs. CHB patients were defined as those who had chronic HBV in-fection with serum HBsAg positivity for �6 months and may have exhib-ited symptoms of hepatitis. The standard for diagnosis of HCC is de-scribed in detail elsewhere (27). In addition, 10 control normal livertissues were obtained from the unused portions of 10 donor livers used forliver transplantation. All patients were hospitalized in the Yantai CityHospital for Infectious Diseases from January 2010 to December 2010.The clinical characteristics of the enrolled subjects are listed in Table 1.Written informed consent was provided by all study participants. Patientsamples were assigned an arbitrary identifier based on the order of enroll-ment in our study. The study protocol was approved by the Ethics Com-mittee of the Yantai City Hospital for Infectious Diseases.

Mice. HBV transgenic BALB/c mice (female, 6 to 8 weeks old) werepurchased from the Transgenic Engineering Lab, Infectious Disease Cen-ter, Guangzhou, China. The HBV transgenic mice were generated with aviral DNA construct, pHBV1.3, containing 1.3 copies of the HBV genome(D genotype). All transgenic mice tested positive for serum HBV surfaceantigen (HBsAg) and virus DNA, as well as the HBV core proteins(HBcAg) in hepatocytes in their livers (28). Animals received humanecare, and the study of mice was in strict accordance with the regulation ofthe Institute of Microbiology, Chinese Academy of Sciences of ResearchEthics Committee. The protocol was approved by the Research EthicsCommittee (permit number PZIMCAS2011001).

Plasmid constructs. The human PTTG1 gene was cloned into thepcDNA3.1 and pEGFP-C1 vectors (Invitrogen), and the recombinantplasmids were named pPTTG1 and pEGFP-PTTG1, respectively. The hu-man PBF gene was cloned into the pcDNA3.1-Myc-His vector, and therecombinant vector was called pPBF. The 3=-untranslated region (UTR)of PBF containing the miR-122 binding site was amplified by PCR usingthe following primers: sense, 5=-GCCCTCGAGCGTAAGGATACGTGGCTTTAGTA-3=; antisense, 5=-CCGGGATCCGTTTTATTGGAAACTGG

TTA-3=. The PCR product was cloned into the XhoI and BamHI sites ofthe pEGFP-C1 vector (pEGFP-PBF-UTR). Mutations were made in theseed region of the miR-122 binding site in the PBF 3=-UTR (pEGFP-PBF-UTRm) to verify the putative miR-122 target site.

Cell culture and transfection. Human hepatoma cell lines (Huh-7,HepG2, and SK-Hep-1) and the L02 immortalized human liver cell linewere obtained from the ATCC (Manassas, VA). A chemically synthesizedmiR-122 inhibitor and nonspecific control, an miR-122 mimic and non-specific control, and a PBF-specific small interfering RNA (siRNA) werepurchased from RiboBio Co., Ltd. (Guangzhou, China). Transfectionswere performed using Lipofectamine 2000 reagent (Invitrogen). Cellswere washed twice with Opti-MEM (Invitrogen) and transfected with 50nM miR-122 inhibitor, miR-122 mimic, PBF siRNA, or 2 �g expressionplasmid. Each treatment was performed at least three times.

RNA extraction and real-time PCR. Total RNA was extracted fromcells or frozen tissues using TRIzol reagent (Invitrogen) according to themanufacturer’s instructions. Real-time fluorescence quantitative PCRwas performed using SYBR green premix reagent (TaKaRa Bio Inc., Shiga,Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was usedas an internal standard for quantification. Real-time PCR analysis formiR-122 was performed using a TaqMan miRNA kit (Applied Biosys-tems). The U6 endogenous control was used for normalization. miR-122primary transcript (pri-miR-122) was detected using a high-capacitycDNA reverse transcription kit (Applied Biosystems) and a TaqMan pri-miRNA assay. Actin was used as an internal standard gene. Relative ex-pression was quantified using the comparative threshold cycle (CT)method.

Luciferase assay. The transcriptional regulation of miR-122 by HBVwas analyzed by transfecting a human hepatoma cell line (Huh-7) withpHBV1.3, pGL-122, or pRL-TK as the control. Each treatment was per-formed at least in triplicate. The pGL-122 construct, with a luciferasereporter gene under the miR-122 promoter, was provided by ShiMeiZhuang (Sun Yat-sen University, China) (8).

Immunohistochemistry (IHC). Liver tissue sections (4 �m thick)were immunostained using a specific rabbit polyclonal antibody to PBF(Santa Cruz Biotech) and an horseradish peroxidase (HRP)-conjugatedsecondary antibody, using 3,3’-diaminobenzidine tetrahydrochloride(DAB) for visualization. PBF staining was assessed according to the fol-lowing scoring approach: nonoverexpression, faint weak staining of thecell cytoplasm; overexpression, moderate to strong cytoplasmic stainingin �80% of hepatocytes.

Western blotting. Western blot analysis was performed as describedpreviously (29).

Northern blotting. Total RNA (30 �g) was subjected to electropho-resis on a 17% Urea-PAGE gel and transferred to a nylon membrane. Themembrane was UV cross-linked and prehybridized at 37°C for 1 h, andthen it was hybridized with a 32P-labeled miR-122 or U6 probe at 37°Covernight. The probe sequence of miR-122 was completely complemen-tary to miR-122 (5=-CAAACACCATTGTCACACTCCA-3=), and the se-quence of the U6 snRNA probe was 5=-GCAGGGGCCATGCTAATCTT

TABLE 1 Clinical characteristics of studied subjects

Parameter

Values by group

HC CHB HCC

Cases (no.) 10 23 19Age (yr; range) 38 (28–53) 43 (30–55) 55 (40–77)Sex (male/female) 7/3 17/6 16/3ALT (U/liter) 21 (15–30) 111 (43–226) 69 (26–190)HBeAg positive (no. [%]) ND 11 (48) 12 (63)HBV viral load (DNA copies/ml serum [range]) ND 9.3 � 106 (1.9 � 105-8.0 � 107) 7.1 � 105 (2.1 � 102-9.8 � 106)HCC stage (I/II/III) ND ND 6/7/6aHCC patient disease stage was evaluated according to the Chinese criterion for diagnosis and staging primary liver cancer instituted by the Chinese Anti-Cancer Association in2001. Abbreviations: HC, healthy control; CHB, chronic hepatitis B; HCC, hepatocellular carcinoma; ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; ND, no data.

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CTCTGTATCG-3=. The membranes were ultimately washed and exposedto PhosphorImager screens (GE Healthcare Bio-Sciences Corp.).

Fluorescence microscopy. Fluorescence microscopy was performedas described previously (29).

CCK-8 assay. The CCK-8 experiment was performed according to ourprevious description (29).

Matrigel invasion assay. The in vitro invasion activity of human hep-atoma cell lines (HepG2, Huh-7, and SK-Hep-1) was measured using atwo-compartment Boyden chamber (Costar) and basement membraneMatrigel. After transfection (48 h), 1 � 105 cells were seeded in the upperchamber of the Transwell insert (8-�m pores) coated with 1 mg/ml Matri-gel (Collaborative Research Inc.). After a 24-h incubation period, thenonmigrating cells in the upper chamber were gently scraped away, andadherent cells present on the lower surface of the insert were stained with4=,6-diamidino-2-phenylindole (DAPI) and photographed by fluores-cence microscopy.

Establishment of stable shRNA transfectant. Three siRNA sequencestargeting the PBF gene were designed. The oligonucleotides were synthe-sized and inserted into the RNA interference (RNAi)-pSIREN-RetroQvector; the short hairpin RNA (shRNA) constructs are described inTable 2. The recombinant plasmid was confirmed by sequencing anddesignated pSIREN-PBFi. Luciferase shRNA was selected as a mock trans-fection control (pSIREN-Luci). Phoenix cells were cotransfected withpSIREN-PBFi or pSIREN-luci and the helper vector. After transfection(48 to 72 h), the supernatant was collected and SK-Hep-1 cells were in-fected with the virus suspension. At 48 h after infection, SK-Hep-1 cellswere placed under puromycin (Ameresco) selection for 2 weeks to estab-lish the stable shRNA cell lines SK-Hep-1-PBFi and SK-Hep-1-Luci. Theprotein levels of PBF were detected by Western blotting to confirm thatthe selected clones contained the shRNA.

Tumor growth in immunodeficient mice. Log-phase SK-Hep-1-PBFiand SK-Hep-1-Luci cells were subcutaneously (s.c.) injected into the rightflank of 5- to 6-week-old female BALB/c-nu/nu mice (1 � 107 cells/mouse, 5 mice/group). The mice were obtained from the Institute of Lab-oratory Animal Science (CAMS&PUMC). Tumor size was then measuredtwice per week. Six weeks after injection, mice were scarified and all tumorxenografts were excised and weighed.

Statistical analysis. Differences between groups were determined us-ing Student’s t test. The degree of association between variables was de-termined by Spearman’s nonparametric correlation. P � 0.05 was consid-ered significant.

RESULTSSuppression of miR-122 by HBV replication. Based on our pre-vious study showing that miR-122 is specifically suppressed inCHB (25), we explored the possible mechanisms underlying HBVinfection-induced miR-122 downregulation. With regard to viralfactors, we tested if HBV expression and replication could affectmiR-122 levels both in vitro and in vivo. Transfection with theHBV replication plasmid pHBV1.3 significantly decreased miR-122 levels in Huh-7 cells (P � 0.01) (Fig. 1A). To confirm the HBVspecificity of downregulation for miR-122, we also examined the

effect of HBV expression on other microRNAs, including miR-21and miR-181a, by real-time PCR. The results demonstrated thatmiR-21 and miR-181a levels were not significantly affected byHBV expression (Fig. 1B). HBV transgenic BALB/c mice werethen used to determine if HBV replication directly affects miR-122levels in vivo. The HBV transgenic mice are generally immunotol-erant to HBV because no liver necroinflammation is observed inthese mice (28), which excludes the possible effect of hepatic in-flammation on miR-122 levels. The HBV transgenic mice werefound to have lower liver miR-122 levels than BALB/c mice (1.0versus 0.675 � 0.06; P � 0.05) (Fig. 1C). We next examined theexpression levels of cyclin G1, a verified miR-122 target (6), in thisanimal model. The results showed that downregulation of miR-

TABLE 2 Oligonucleotide sequences of PBF shRNAs

PBFshRNA no. Sense Antisense

1 5=-GATCCGGAGCTGCTTGTTCTCAGATTCAAGAGATCTGAGAACAAGCAGCTCCTTTTTTG-3=

5=-AATTCAAAAAAGGAGCTGCTTGTTCTCAGATCTCTTGAATCTGAGAACAAGCAGCTCCG-3=

2 5=-GATCCCCGGCTTCCCTTTGTAAATTTCAAGAGAATTTACAAAGGGAAGCCGGTTTTTTG-3

5=-AATTCAAAAAACCGGCTTCCCTTTGTAAATTCTCTTGAAATTTACAAAGGGAAGCCGGG-3=

3 5=-GATCCCCGTATGCTAGATTTGAAATTCAAGAGATTTCAAATCTAGCATACGGTTTTTTG-3=

5=-AATTCAAAAAACCGTATGCTAGATTTGAAATCTCTTGAATTTCAAATCTAGCATACGGG-3=

FIG 1 Suppression of miR-122 by HBV replication. (A and B) After transfec-tion with the HBV replication vector pHBV1.3 or empty pcDNA3.1 plasmid(mock), miR-122 expression (A) and the expression of miR-21 and miR-181a(B) in Huh-7 cells were quantified by real-time PCR. The results were normal-ized to a U6 endogenous control, RNU6B. (C and D) Detection of miR-122expression (C) and cyclin G1 expression (D) in the liver of HBV transgenicBALB/c mice (n � 5) and control BALB/c mice (n � 5) by real-time PCR. P �0.05 (*) and P � 0.01 (**) compared to the control or mock treatment.

miR-122 Inhibition by HBV mRNAs Upregulates PBF

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FIG 2 Inhibition of miR-122 by HBV mRNAs. (A) miR-122 promoter activity in response to pHBV1.3 transfection. Huh-7 cells were cotransfected withpHBV1.3 or the empty pcDNA3.1 vector (mock transfection), pGL-122 with a luciferase reporter under the miR-122 promoter, and pRL-TK. pGL-122 andrenilla luciferase activities were measured using a dual-luciferase assay kit at the indicated time. (B) After transfection with pHBV1.3 or pcDNA3.1, pri-miR-122expression in Huh-7 cells was quantified with real-time PCR. (C) Detection of the mRNA levels of miR-122 transcription factors after transfection with pHBV1.3or pcDNA3.1 for 72 h in Huh-7 cells. (D, top) Predicted miR-122 binding sequence located in the HBV genome at nt 1684 to 1709. Perfect matches are indicatedby a line. Mutations were made in the seed region of the miR-122 binding sites (HBV-mut) without changing the amino acid sequence of the X protein. (Lower)A putative miR-122 complementary region was found in all four HBV mRNAs. 293T cells were cotransfected with an miR-122 mimic or a randomizedoligonucleotide (mock transfection) and a firefly luciferase reporter plasmid with a 3=-UTR containing either the wild-type (HBV-wt) or mutant (HBV-mut)

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122 in the HBV transgenic mice caused an increase in cyclin G1levels (Fig. 1D). Taken together, the results suggest that the de-crease in miR-122 in chronic HBV infection is attributed to viralreplication.

HBV mRNAs with a complementary binding site to miR-122sequester endogenous miR-122. To understand how HBV repli-cation downregulates miR-122 levels, we first tested if the viralfactors influence miR-122 transcription. A luciferase reporter as-say was performed using the pGL-122 construct containing aluciferase reporter gene under the miR-122 promoter (8). Unex-pectedly, transfection with pHBV1.3 caused an increase in miR-122 promoter activity at all time points (Fig. 2A). Real-time PCRanalysis also revealed an increase in miR-122 primary transcript(pri-miR-122) levels after pHBV1.3 treatment (Fig. 2B), validat-ing that viral replication actually enhances miR-122 transcription.We then examined the expression levels of miR-122 transcriptionfactors, including HNF1�, HNF4�, HNF3, and C/EBP� (30). Asshown in Fig. 2C, HNF4� and C/EBP� expression levels increasedafter transfection with pHBV1.3.

The data described above indicate that transfection withpHBV1.3 led to a significant decrease in mature miR-122 levelsbut an obvious increase in miR-122 transcription. To explore thisdiscrepancy, we hypothesized that the HBV-induced decrease ofmiR-122 occurs posttranscriptionally. Because the miRNAsponge method, i.e., targeting an miRNA of interest with a spongetransgene having multiple complementary miRNA sites, has beenwidely used for miRNA inhibition studies (31), we searched forputative miR-122 binding sites in the HBV genomic sequenceusing Miranda software. A putative miR-122 complementary re-gion (spanning nucleotides 1684 to 1709 in the HBV genome) wasfound in all four HBV mRNAs, including the pre-C/C (orpgRNA), pre-S, S 3=-UTR, and X mRNAs (Fig. 2D). Mutationswere made in the seed region of the miR-122 binding site as acontrol. This miR-122 binding site is highly conserved amongdifferent HBV genotypes (data not shown). Treatment with anmiR-122 mimic reduced the reporter activity of the wild-type(HBV-wt) but not mutant (HBV-mut) HBV 3=-UTR-containingluciferase reporter (Fig. 2D).

To determine if the miR-122 binding site in the HBV 3=-UTRcan act as a sponge to inhibit miR-122, Huh-7 cells with constitu-tive high expression of miR-122 were transfected with an HBV3=-UTR-containing luciferase reporter. As shown in Fig. 2E, com-pared to the mock control, transfection of the wild-type 3=-UTRbut not the mutant 3=-UTR led to decreased miR-122 levels (P �0.05). To confirm that the miR-122 binding site in the HBV genecan lead to miR-122 inhibition, the HBV replication vectorpHBV1.3 was mutated within the miR-122 binding seed region asshown in Fig. 2D. Both real-time PCR and Northern blotting re-sults demonstrated that transfection of wild-type but not mutantHBV plasmid resulted in a sharp decrease in the miR-122 levels(Fig. 2F), validating that the miR-122 binding sites in HBV

mRNAs sequester endogenous miR-122. The depletion of miR-122 by HBV was further confirmed by Western blotting of theexpression of the miR-122 target, cyclin G1 (Fig. 2G). To furtherconfirm that the decreased miR-122 is the result of direct seques-tration of miR-122 by HBV mRNAs, the YMDD motif in the poly-merase sequence of pHBV1.3 was modified to YMHD by substi-tuting aspartic acid at position 540 for histidine, and the constructwas named pYMHD1.3. We found that pYMHD1.3 could stillinhibit miR-122 (Fig. 2H). Because YMHD instead of YMDD inthe polymerase results in reduced reverse transcriptase activityand HBV replication (32), these data suggest that HBV mRNA candirectly sequester miR-122. Furthermore, the decrease of miR-122followed a dose-response curve of transfection with wild-typeHBV plasmid, and it was synchronized with the increase of totalHBV mRNA levels (Fig. 2I). Interestingly, transfection with themutant HBV plasmid led to increased miR-122 levels compared tothose of the empty plasmid control (Fig. 2F), which is consistentwith the observations (Fig. 2A and 2B) that viral factors actuallyenhance miR-122 transcription.

We further examined the association between miR-122 levelsand intrahepatic total HBV mRNA levels in CHB patients. As seenin Fig. 2J, there was a negative correlation between miR-122 levelsand total viral mRNA levels, which provides further evidence thatviral mRNAs lead to miR-122 suppression. Collectively, these re-sults suggest that the decline in miR-122 levels induced by HBVexpression and replication occurs via saturation and depletion ofmiR-122 via binding to the complementary binding sites locatedin HBV mRNAs.

Meanwhile, another putative miR-122 target site was alsofound in the viral pregenomic RNA (spanning nucleotides 2738 to2760 in the HBV genome), but transfection of the luciferase re-porter with a 3=-UTR containing this region did not lead to de-creased miR-122 levels. Further, there was no significant differ-ence of miR-122 levels in Huh-7 cells transfected with wild-type ormutant HBV plasmids (data not shown).

Inhibition of miR-122 leads to increased expression of itstarget PBF. Because distinctive downregulation of miR-122 wasobserved in liver tissues of CHB and HCC patients (0.3541 �0.176 [CHB] versus 1 � 0.0721 [HC], P � 0.014; 0.0821 � 0.0286[HCC] versus 1 � 0.0721 [HC], P � 0.00058), and HCC patientshad lower miR-122 levels than CHB patients (0.3541 � 0.176[CHB] versus 0.0821 � 0.0286 [HCC], P � 0.11) (Fig. 3A), wenext investigated the miR-122 targets that may play a role in CHB-induced HCC. The 3=-UTR of PBF was identified as a predictedtarget of miR-122. A putative miR-122 complementary region inthe PTTG1IP sequence (position 2202 to 2223 in the PTTG1IP3=-UTR) is shown in Fig. 3B. We found that miR-122 inhibitedgreen fluorescent protein (GFP) expression of a wild-type (EGFP-PBF-UTR) but not mutant 3=-UTR (EGFP-PBF-UTRm) of PBFin a GFP reporter assay. HepG2, Huh-7, L02, and SK-Hep-1 cellstransfected with the miR-122 mimic displayed a decrease in PBF

miR-122 binding sequence. The firefly luciferase and renilla luciferase activities were measured using a dual-luciferase assay kit. (E) Huh-7 cells were transfectedwith the luciferase reporter plasmid with either the HBV-wt or HBV-mut 3=-UTR, and miR-122 levels were measured by real-time PCR 24 h after transfection.(F and G) Huh-7 cells were transfected with the wild-type (pHBV1.3-wt) or mutant (pHBV1.3-mut) HBV plasmid or with pcDNA3.1 (mock transfection). (F)At 24 h after transfection, miR-122 levels were assessed by real-time PCR (left) and Northern blotting (right). (G) The expression of cyclin G1 was detected at 48h by Western blotting. (H) Huh-7 cells were transfected with pYMHD1.3 or pcDNA3.1, and at 24 h after transfection miR-122 levels were measured by real-timePCR. (I) Huh-7 cells were transfected with the indicated amount of pHBV1.3, and miR-122 levels and total HBV mRNA levels were analyzed by real-time PCR.(J) Correlation analysis between miR-122 levels and intrahepatic HBV mRNA levels in CHB by Spearman analysis. Values of the correlation coefficient (R) andP are shown. P � 0.05 (*) and P � 0.01 (**) compared to mock transfection.

miR-122 Inhibition by HBV mRNAs Upregulates PBF

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expression, while inhibition of endogenous miR-122 in Huh-7cells by the miR-122 inhibitor resulted in an increase of PBF (Fig.3C). As expected, transfection of the HBV plasmid caused an ob-vious increase in PBF levels in Huh-7 cells (Fig. 3D).

We further examined the PBF levels in the livers of these pa-tients by IHC analysis (Fig. 3E). Overexpression of PBF was ob-served in 39% of CHB (9 of 23), 68% of HCC (13 of 19), and nohealthy individuals (HC) (0/10). A negative relationship betweenmiR-122 levels and PBF overexpression was found in CHB pa-tients (P � 0.05) (Fig. 3F). Together, these data suggest that miR-122 downregulation induced by CHB elevates the expression of itstarget PBF.

PBF increases liver cancer cell proliferation and invasiveability and promotes tumor growth in nude mice. We next ex-plored the possibility that PBF upregulation in CHB can contrib-ute to the increased growth potential of hepatoma cells. As seen inFig. 4A, knockdown of PBF expression by siRNA reduced Huh-7cell proliferation, whereas overexpression of PBF after transfec-tion of the pPBF expression vector resulted in increased cell pro-liferation (P � 0.001 or P � 0.01). Similar results were observed inHepG2 (Fig. 4B) and SK-Hep-1 cells (Fig. 4C). Subsequently, westudied whether miR-122 inhibits cell proliferation through PBF.Transfection with an miR-122 mimic significantly reduced cellproliferation in HepG2 cells but not in cells treated with PBF

FIG 3 PBF, an miR-122 target, is upregulated in patients with CHB and HCC. (A) Detection of miR-122 expression in liver biopsy specimens from CHB, HCC(the tumor), and healthy controls (HC) by real-time PCR. The results were normalized to a U6 endogenous control, RNU6B. The miR-122 levels in HC werearbitrarily set to 1.0. (B, top) Perfect matches are indicated by a line between the 3=-UTR of PBF (PBF-UTR) and miR-122. Mutations (PBF-UTRm) were madein the seed region of the miR-122 binding site for the reporter gene assay. (Lower) HepG2 cells were cotransfected with an miR-122 mimic or a randomizedoligonucleotide as a mock transfection and an EGFP reporter plasmid carrying either the wild-type (EGFP-PBF-UTR) or mutant (EGFP-PBF-UTRm) 3=-UTRof PBF. The GFP levels were measured by Western blotting. (C and D) Western blot analysis of the expression of PBF in HepG2, SK-Hep-1, Huh-7, and L02 cellstransfected with an miR-122 mimic or inhibitor (C) or transfected with the wild-type (pHBV1.3-wt) and mutant (pHBV1.3-mut) HBV replication vector (D).(E) Immunohistochemistry analysis of PBF expression in the liver samples from CHB, HCC, and HC. (F) Distribution of the miR-122 levels in CHB patients withPBF overexpression and nonoverexpression in liver tissue. Data are expressed as the medians and ranges (from the 10th percentile to the 90th percentile). *, P �0.05.

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siRNA (Fig. 4D). Moreover, depletion of miR-122 by its inhibitorled to a significant increase in cell proliferation in Huh-7 cells butnot in PBF siRNA-treated cells (Fig. 4E). The results indicate thatmiR-122 at least partially suppresses cell proliferation throughdownregulation of PBF. We also found that pHBV1.3-wt trans-fection increased Huh-7 cell proliferation (Fig. 4F), whereas trans-fection with pHBV1.3-mut with mutations in the miR-122 bind-

ing site did not (Fig. 4G). Notably, the growth promotion effect ofHBV on Huh-7 cells was almost abolished by cotransfection ofmiR-122 (Fig. 4H) or PBF siRNA (Fig. 4I). These results suggestthat HBV promotes cell proliferation by inhibition of miR-122and the subsequent upregulation of PBF.

We next examined the effect of PBF on cell invasion using aMatrigel invasion assay (Fig. 5A). As shown in Fig. 5B, compared

FIG 4 PBF enhances the growth of liver cancer cells. (A to C) Huh-7 (A), HepG2 (B), and SK-Hep-1 (C) cells were transfected with PBF siRNA (or the controlsiRNA as a mock transfection) or with the PBF expression vector pPBF (or pcDNA3.1-Myc-his as a mock transfection). At 24, 48, and 72 h after transfection, cellgrowth was assessed by CCK-8 assay. (D and E) HepG2 cells were transfected with an miR-122 mimic or a randomized oligonucleotide as a mock transfectionand PBF siRNA (D), and Huh-7 cells were transfected with an miR-122 inhibitor or a randomized oligonucleotide and PBF siRNA (E). At 24, 48, and 72 h aftertransfection, cell growth was measured by CCK-8 assay. (F to I) Huh-7 cells were transfected with pHBV1.3-wt or pcDNA3.1 (F) or with pHBV1.3-mut orpcDNA3.1 (G), or they were cotransfected with pHBV1.3 or pcDNA3.1, along with an miR-122 mimic (H) or PBF siRNA (I). At 24, 48, and 72 h aftertransfection, cell growth was measured by CCK-8 assay. P � 0.05 (*), P � 0.01 (**), and P � 0.001 (***) compared to mock transfection.

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to the mock transfection, overexpression of PBF or PTTG1 bytransfection with the pPBF or pPTTG1 vector in Huh-7, HepG2,and SK-Hep-1 cells led to significantly increased invasion throughMatrigel-coated membranes. Notably, the invasion activity was

significantly enhanced in cells cotransfected with pPBF andpPTTG1 compared to transfection with either alone. We thenexamined if HBV also increases cell invasion. Our results showthat transfection with pHBV1.3-wt (Fig. 5C) but not pHBV1.3-

FIG 5 PBF enhances the invasion of liver cancer cells. (A) Huh-7, HepG2, and SK-Hep-1 cells were transfected with pPBF and/or pPTTG1 or pcDNA3.1-Myc-his (mocktransfection). The cell invasion activity was then assessed using a Matrigel-coated Boyden chamber assay. Representative images of invading cells (stained with DAPI)were photographed with a fluorescence microscope. (B) The mean numbers of invading cells are shown; results are presented as means � SD from three independentexperiments. (C and D) Huh-7 cells were transfected with pHBV1.3-wt or pcDNA3.1 (C) or with pHBV1.3-mut or pcDNA3.1 (D). The cell invasion ability was thenmeasured, and the mean numbers of invading cells are shown. (E and F) Huh-7 cells were cotransfected with pHBV1.3 or pcDNA3.1, along with an miR-122 mimic (E)or PBF siRNA (F). The mean numbers of invading cells are shown. P � 0.05 (*), P � 0.01 (**), and P � 0.001 (***) compared to the mock transfection.

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mut (Fig. 5D) in Huh-7 cells increased cell invasion activity.Moreover, the effect of HBV on cell invasion was largely abolishedby simultaneous transfection of miR-122 (Fig. 5E) or PBF siRNA(Fig. 5F), indicating that HBV promotes invasion by decreasingmiR-122 and increasing PBF.

Additionally, we examined the growth-promoting function ofPBF by generating a stable PBF knockdown SK-Hep-1 cell line,SK-Hep-1-PBFi (Fig. 6A). In concert with the in vitro results de-scribed above, tumor growth was significantly slowed in SK-Hep-1-PBFi-xenografted nude mice compared to that in mock-treatedmice (Fig. 6B). PBF depletion in SK-Hep-1 cells resulted in de-creased tumor weight by 65.6% (P � 0.05) (Fig. 6C). Depletion ofPBF by RNAi in tumors was verified by IHC detection (Fig. 6D).Together, these in vitro and in vivo results demonstrate the tumor-promoting properties of PBF in HCC cells and suggest that the lossof miR-122 by HBV expression and subsequent upregulation of itstarget PBF in CHB contribute to the development of HCC.

PBF interacts with PTTG1 and promotes its transcriptionalactivities by facilitating PTTG1 nuclear translocation. It isknown that PBF is associated with PTTG1, which is a proto-on-cogene in human endocrine cancer (33, 34). Thus, we examinedthe effect of PBF on the subcellular localization of PTTG1. As seenin Fig. 7A, in HepG2 and Huh-7 cells transfected with the pEGFP-PTTG1 expression vector, PTTG1 localized to both the nucleusand cytoplasm by immunofluorescence (first row), but it localizedto the nucleus when cotransfected with the pPBF expression vec-tor (second row). To further assess if the interaction between PBFand PTTG1 affects the function of PTTG1 as a transcriptionalactivator, we performed real-time PCR analysis of the expressionof its target genes VEGF, FGF-2, c-myc, and MMP-2 (35). Asshown in Fig. 7B, compared to mock treatment, the mRNA levelsof PTTG1 transcriptional target genes significantly increased inpPBF-transfected cells and decreased in PBF siRNA-treated cells.

Together, these results demonstrate that PBF promotes PTTG1transcriptional activities by facilitating PTTG1 translocation intothe nucleus.

DISCUSSION

HBV infection-associated carcinogenesis is a multifactorial andmultistep process with many etiologies, and it involves a variety ofgenetic and epigenetic alterations as well as deregulation of vari-ous signaling pathways over the long latency period of tumor for-mation. However, the molecular mechanisms underlying CHB-induced HCC remain largely debated (14–16). In the presentstudy, we investigated the clinical and functional relevance of thehighly expressed HBV mRNAs and liver-specific miR-122 for thedevelopment of HCC in HBV infection. We found that all fourHBV mRNAs contain a binding site that is complementary tomiR-122 and may act as a sponge to bind and sequester endoge-nous miR-122, contributing to HBV-induced miR-122 inhibi-tion. In addition, we demonstrated that downregulation of miR-122 leads to increased expression of its target PBF, thus promotingthe transcriptional activities of PTTG1 by facilitating its nucleartranslocation. This in turn promotes HCC cell growth and inva-sion and in vivo tumor growth. Consistent with our results, severalother studies (36, 37) also show that miR-122 is decreased in cellsthat produce HBV, but the mechanism by which this occurs isunknown. Therefore, our results provide the first insights intomiR-122 inhibition by HBV mRNA and demonstrate the mecha-nism of HBV-mediated cell growth and invasion by activation ofPTTG1 through upregulation of the miR-122 target PBF, asshown in Fig. 7C. According to this model, loss of miR-122 bysequestration with HBV mRNAs may contribute to the develop-ment of HCC.

The major contribution of this work is to demonstrate that anmiR-122 binding site exists in all four HBV mRNAs and that dur-

FIG 6 Effect of PBF knockdown on liver tumor growth in nude mice. (A) Western blot analysis of PBF expression in SK-Hep-1 cells stably transfected with PBFsiRNA (SK-Hep-1-PBFi) and luciferase siRNA-transfected cells (SK-Hep-1-Luci) as a mock transfection. Actin was used as a loading control. (B) SK-Hep-1-PBFi(PBF siRNA) or SK-Hep-1-Luci (mock) cells were s.c. injected into female BALB/c-nu/nu mice. Tumor diameters were measured twice a week for 6 weeks. (C)Tumor weight was measured when the mice were sacrificed at week 6. The results are presented as means � SD from five mice. (D) Paraffin tumor tissue sectionswere stained with anti-PBF polyclonal antibody and counterstained with hematoxylin. *, P � 0.05 compared to mock treatment. Data are representative of twoindependent experiments.

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ing HBV infection, these miR-122 target mRNAs can sequesterendogenous miR-122 in hepatocytes, possibly contributing to thedecrease in miR-122 and upregulation of its target genes, e.g., PBFand cyclin G1 (Fig. 7D). To our knowledge, this is the first reportof such miRNA sequestration and depletion by viral mRNAs inany infection.

The observation that transfection of an HBV plasmid enhancesmiR-122 transcription but results in a decrease of mature miR-122 led us to speculate that miR-122 could be sequestered by viralmRNAs, similar to how the miRNA sponge method has been used

for miRNA inhibition studies by expressing exogenous spongeRNAs with multiple sites complementary to the miRNA of inter-est (31, 38). This hypothesis was supported by our finding thattransfection of the luciferase reporter with the wild-type HBV3=-UTR or an HBV replication plasmid harboring the miR-122binding site (nt 1684 to 1709) led to decreased miR-122 levels,while transfection of mutant 3=-UTR or HBV plasmid did not(Fig. 2E and F). In addition, miR-122 levels were negatively asso-ciated with viral mRNA levels (Fig. 2I and J), validating that themiR-122 binding sites in the HBV mRNAs sequester endogenous

FIG 7 PBF promotes PTTG1 transcriptional activity by facilitating PTTG1 nuclear translocation. (A) HepG2 and Huh-7 cells were cotransfected with pEGFP-PTTG1 and pPBF or the empty pcDNA3.1-Myc-His vector as a mock transfection. At 48 h after transfection, cells were fixed and PTTG1 was directly detectedby green fluorescence. DAPI staining (blue) indicates the nucleus. (B) HepG2 cells were transfected with pPBF (or pcDNA3.1-Myc-his) or PBF siRNA (or controlsiRNA). At 48 h after transfection, the mRNA levels of VEGF, FGF-2, Sp1, c-myc, and MMP-2 were measured by real-time PCR. The data are presented as themean ratios (� SD) compared to those for mock transfection. Results are presented from three independent experiments. P � 0.05 (*), P � 0.01 (**), and P �0.001 (***) compared to the mock transfection. (C) Schematic of how HBV-miR-122-PBF-PTTG1 may mediate liver cell growth and invasion. Inhibition (�)or stimulation (2) was determined according to how CHB affects miR-122 expression and how miR-122 may contribute to HCC development. In CHB patients,miR-122 in hepatocytes is inhibited by viral mRNAs and/or chronic inflammation. Loss of miR-122 expression leads to upregulation of its target PBF, whichinitiates PTTG1 nuclear translocation, promoting PTTG1 transcriptional activity and thus enhancing cell growth and invasion. (D) HBV mRNAs act as spongesto block miR-122-mediated inhibition of endogenous target genes. In hepatocytes with no HBV infection, target mRNAs of miR-122 (black) are repressed by themiR-122/RISC complex (green rectangles). miR-122-induced target mRNA degradation is shown by black dashed lines. After HBV infection, viral mRNAs (red)are expressed at a high level and sequester the miR-122 complexes, rescuing the expression of target mRNAs.

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miR-122. These results are consistent with our previous studyshowing that miR-122 levels are negatively correlated with intra-hepatic HBV load in CHB (25). Several lines of evidence supportour hypothesis. Previous studies (36, 37) have shown that miR-122 is downregulated in the HBV-expressing HCC cell line. Cou-louarn et al. (26) showed that miR-122 is repressed in HBV-in-fected but not HCV-infected HCC, indicating that the expressionof miR-122 is etiology dependent. A similar result was also re-ported by Sarasin-Filipowicz and colleagues, i.e., that decreasedmiR-122 levels are observed in only a small portion of HCV pa-tients (39), which is in contrast to the observation that almost allCHB patients have much lower miR-122 levels than healthy con-trols. Of note, we demonstrated that miR-122 levels could be sig-nificantly decreased by HBV expression both in vitro and in vivo,whereas no obvious change in miR-122 levels was observed inHCV-infected Huh7.5 cells (40). However, aside from HBVmRNAs, the HCV RNA 5=-UTR can also bind to miR-122 (11, 13).We speculate that this discrepancy is due to the different viralRNA abundances between HBV and HCV, because based on pre-vious studies, the HBV RNA copy number per cell reaches 105 (25,41), whereas for HCV, the RNA copy number is only thousandsper cell (42). Considering that miR-122 is expressed in hepato-cytes at �50,000 copies per cell (43) and the abundant HBV tran-scription from viral covalently closed circular DNA (cccDNA) inhepatocytes, it is conceivable that the large amounts of viralmRNAs harboring the miR-122 binding site exert their suppres-sion of miR-122 levels as miR-122 sponges. Besides the miR-122binding sites (nt 1684 to 1709) that we identified in all four HBVmRNAs, another miR-122 binding site in the viral pgRNA (nt2738 to 2760) has been found to be a target for miR-122-inducedinhibition of HBV gene expression (36). However, our studyshowed that this miR-122 binding site in pgRNA does not lead tosignificant inhibition of miR-122. This may be due to the smallproportion of pgRNA relative to the total viral mRNAs. This de-serves further investigation.

Interestingly, we also found that direct targeting of HBVmRNAs only plays a minor role in miR-122-mediated inhibitionof HBV replication (data not shown), which is consistent with ourprevious study showing that miR-122 mainly suppresses HBVreplication via activation of p53 (25). A possible interpretation isthat highly redundant HBV transcripts are produced from viralcccDNA in the hepatocytes (44). Thus, it is possible that the po-tential impact of miR-122 on HBV replication is partly masked byviral mRNA sponges, as a similar phenomenon was recently ob-served in the recombinant coxsackievirus, showing that the virusmay overcome host microRNA-induced inhibition by microRNAsaturation (30). Conceivably, the interaction between miR-122and viral mRNAs has a reciprocal effect on HBV RNA levels. Tar-geting of HBV mRNAs by miR-122 may lead to degradation andtranslational repression of the target mRNAs. Meanwhile, the sat-uration and depletion of miR-122 via binding to HBV mRNAsincreases its target cyclin G1, which may enhance HBV transcrip-tion and expression by the p53 pathway (25). The mechanismunderlying the miR-122-mediated regulation of viral mRNAs isunder investigation.

To date, PBF is a relatively uncharacterized oncoprotein whosefunction remains largely unknown, although it is ubiquitouslyexpressed and highly conserved across a wide diversity of animalspecies. PBF is overexpressed in pituitary, thyroid, and breast can-cer and is associated with poor prognosis (35, 45, 46), but it has

not been studied in the context of HCC. Interestingly, increasedexpression of PTTG1 is observed as CHB progresses to cirrhosisand HCC (47). Conceivably, PBF is not the only target of miR-122that is relevant to CHB-induced HCC. A previous study showedthat miR-122 plays an important role in HBV-related hepatocar-cinogenesis by targeting NDRG3, a member of the N-myc down-stream-regulated gene (NDRG) family (37). In addition, we pre-viously also found that the miR-122 target cyclin G1 isoverexpressed in CHB (25). Nevertheless, in this study the pro-moting effect of HBV on cellular proliferation and invasion wasshown to be largely dependent on the regulation of miR-122 andPBF (Fig. 4 and 5). It will be worthwhile to investigate how othermechanisms contribute to tumorigenesis via miR-122 targets,such as ADAM17 (7), SRF (5), Igf1R (8), and cyclin G1 (6), inaddition to NDRG3, and whether PBF overexpression is a com-mon feature of HBV-associated HCC.

Finally, we observed lower miR-122 levels in HBV-infectedHCC than in CHB, which cannot solely be attributed to HBVmRNA-mediated miR-122 sequestration. Decreased miR-122 ex-pression in both HBV-infected and -uninfected HCC, which isassociated with poor prognosis and metastasis, has been con-firmed by several other studies (5, 7, 8, 48). Thus, mechanisticstudies are urgently needed to explore the causes of reduction ofmiR-122 in HCC.

To conclude, our current study provides novel insights into themechanism of miR-122 saturation and depletion by binding to thecomplementary sites located in HBV mRNAs. Furthermore, ourdata suggest that downregulation of miR-122 during chronic HBVinfection induces increased expression of its target PBF, whichimproves our understanding of the molecular mechanisms ofHBV-related carcinogenesis. Therefore, our work demonstratesthe reciprocal interplay of host miRNA sequestration and deple-tion by viral mRNAs. Given the broad interactions of miRNAs andtheir target mRNAs in viral infections, our data may expand theknowledge of how viral factors modulate host cell signaling path-ways by multiple mechanisms and provide a rationale for the de-velopment of novel therapies to prevent the development of can-cer under chronic viral infection.

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural ScienceFoundation of China (91029724, 31230026, 30970146, 81021003, and81102018), grants from the Major State Basic Research DevelopmentProgram (no. 2011CB504802 and 2012CB519003), and a grant fromKey Projects in the National Science & Technology Program(2012ZX10004501-001-003).

REFERENCES1. Krol J, Loedige I, Filipowicz W. 2010. The widespread regulation of

microRNA biogenesis, function and decay. Nat. Rev. Genet. 11:597– 610.2. Girard M, Jacquemin E, Munnich A, Lyonnet S, Henrion-Caude A.

2008. miR-122, a paradigm for the role of microRNAs in the liver. J.Hepatol. 48:648 – 656.

3. Castoldi M, Vujic Spasic M, Altamura S, Elmén J, Lindow M, Kiss J,Stolte J, Sparla R, D’Alessandro LA, Klingmüller U, Fleming RE,Longerich T, Gröne HJ, Benes V, Kauppinen S, Hentze MW, Muck-enthaler MU. 2011. The liver-specific microRNA miR-122 controls sys-temic iron homeostasis in mice. J. Clin. Investig. 121:1386 –1396.

4. Xu H, He JH, Xiao ZD, Zhang QQ, Chen YQ, Zhou H, Qu LH. 2010.Liver-enriched transcription factors regulate microRNA-122 that targetsCUTL1 during liver development. Hepatology 52:1431–1442.

5. Bai S, Nasser MW, Wang B, Hsu SH, Datta J, Kutay H, Yadav A, YadavA, Nuovo G, Kumar P, Ghoshal K. 2009. MicroRNA-122 inhibits tu-

miR-122 Inhibition by HBV mRNAs Upregulates PBF

February 2013 Volume 87 Number 4 jvi.asm.org 2203

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Nov

embe

r 20

21 b

y 77

.38.

223.

73.

morigenic properties of hepatocellular carcinoma cells and sensitizes thesecells to sorafenib. J. Biol. Chem. 284:32015–32027.

6. Fornari F, Gramantieri L, Giovannini C, Veronese A, Ferracin M,Sabbioni S, Calin GA, Grazi GL, Croce CM, Tavolari S, Chieco P,Negrini M, Bolondi L. 2009. miR-122/cyclin G1 interaction modulatesp53 activity and affects doxorubicin sensitivity of human hepatocarci-noma cells. Cancer Res. 69:5761–5767.

7. Tsai WC, Hsu PW, Lai TC, Chau GY, Lin CW, Chen CM, Lin CD,Liao YL, Wang JL, Chau YP, Hsu MT, Hsiao M, Huang HD, TsouAP. 2009. MicroRNA-122, a tumor suppressor microRNA that regu-lates intrahepatic metastasis of hepatocellular carcinoma. Hepatology49:1571–1582.

8. Zeng C, Wang R, Li D, Lin XJ, Wei QK, Yuan Y, Wang Q, Chen W,Zhuang SM. 2010. A novel GSK-3 beta-C/EBP alpha-miR-122-insulin-like growth factor 1 receptor regulatory circuitry in human hepatocellularcarcinoma. Hepatology 52:1702–1712.

9. Norman KL, Sarnow P. 2010. Modulation of hepatitis C virus RNAabundance and the isoprenoid biosynthesis pathway by microRNA miR-122 involves distinct mechanisms. J. Virol. 84:666 – 670.

10. O’Connell RM, Rao DS, Chaudhuri AA, Baltimore D. 2010. Physiolog-ical and pathological roles for microRNAs in the immune system. Nat.Rev. Immunol. 10:111–122.

11. Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M,Munk ME, Kauppinen S, Orum H. 2010. Therapeutic silencing of mi-croRNA-122 in primates with chronic hepatitis C virus infection. Science327:198 –201.

12. Qiu L, Fan H, Jin W, Zhao B, Wang Y, Ju Y, Chen L, Chen Y, Duan Z,Meng S. 2010. miR-122-induced down-regulation of HO-1 negativelyaffects miR-122-mediated suppression of HBV. Biochem. Biophys. Res.Commun. 398:771–777.

13. Shimakami T, Yamane D, Jangra RK, Kempf BJ, Spaniel C, Barton DJ,Lemon SM. 2012. Stabilization of hepatitis C virus RNA by an Ago2-miR-122 complex. Proc. Natl. Acad. Sci. U. S. A. 10:941–946.

14. De Mitri MS, Cassini R, Bernardi M. 2010. Hepatitis B virus-relatedhepatocarcinogenesis: molecular oncogenic potential of clear or occultinfections. Eur. J. Cancer 46:2178 –2186.

15. Neuveut C, Wei Y, Buendia MA. 2010. Mechanisms of HBV-relatedhepatocarcinogenesis. J. Hepatol. 52:594 – 604.

16. Rehermann B, Nascimbeni M. 2005. Immunology of hepatitis B virusand hepatitis C virus infection. Nat. Rev. Immunol. 5:215–229.

17. Beasley RP, Hwang LY, Lin CC, Chien CS. 1981. Hepatocellular carci-noma and hepatitis B virus. A prospective study of 22707 men in Taiwan.Lancet ii:1129 –1133.

18. Kew MC. 2010. Epidemiology of chronic hepatitis B virus infection, hep-atocellular carcinoma, and hepatitis B virus-induced hepatocellular carci-noma. Pathol. Biol. (Paris) 58:273–277.

19. Wang WH, Studach LL, Andrisani OM. 2011. Proteins ZNF198 andSUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediatedhepatocyte transformation and in HBV replication. Hepatology 53:1137–1147.

20. Yang HI, Yeh SH, Chen PJ, Iloeje UH, Jen CL, Su J, Wang LY, LuSN, You SL, Chen DS, Liaw YF, Chen CJ, Study Group REVEAL-HBV. 2008. Associations between hepatitis B virus genotype and mu-tants and the risk of hepatocellular carcinoma. J. Natl. Cancer Inst.100:1134 –1143.

21. Barash H, Gross RE, Edrei Y, Ella E, Israel A, Cohen I, Corchia N,Ben-Moshe T, Pappo O, Pikarsky E, Goldenberg D, Shiloh Y, Galun E,Abramovitch R. 2010. Accelerated carcinogenesis following liver regen-eration is associated with chronic inflammation-induced double-strandDNA breaks. Proc. Natl. Acad. Sci. U. S. A. 107:2207–2212.

22. Yang JC, Teng CF, Wu HC, Tsai HW, Chuang HC, Tsai TF, Hsu YH,Huang W, Wu LW, Su IJ. 2009. Enhanced expression of vascular endo-thelial growth factor-A in ground glass hepatocytes and its implication inhepatitis B virus hepatocarcinogenesis. Hepatology 49:1962–1971.

23. Zhao J, Wu G, Bu F, Lu B, Liang A, Cao L, Tong X, Lu X, Wu M, GuoY. 2010. Epigenetic silence of ankyrin-repeat-containing, SH3-domain-containing, and proline-rich-region-containing protein 1 (ASPP1) andASPP2 genes promotes tumor growth in hepatitis B virus-positive hepa-tocellular carcinoma. Hepatology 51:142–153.

24. Zhang H, Zhai Y, Hu Z, Wu C, Qian J, Jia W, Ma F, Huang W, Yu L,Yue W, Wang Z, Li P, Zhang Y, Liang R, Wei Z, Cui Y, Xie W, Cai M,Yu X, Yuan Y, Xia X, Zhang X, Yang H, Qiu W, Yang J, Gong F, ChenM, Shen H, Lin D, Zeng YX, He F, Zhou G. 2010. Genome-wide

association study identifies 1p36.22 as a new susceptibility locus for hep-atocellular carcinoma in chronic hepatitis B virus carriers. Nat. Genet.42:755–758.

25. Wang S, Qiu L, Yan X, Jin W, Wang Y, Chen L, Wu E, Ye X, Gao GF,Wang F, Chen Y, Duan Z, Meng S. 2012. Loss of miR-122 expression inpatients with hepatitis B enhances hepatitis B virus replication throughcyclin G1 modulated P53 activity. Hepatology 55:730 –741.

26. Coulouarn C, Factor VM, Andersen JB, Durkin ME, Thorgeirsson SS.2009. Loss of miR-122 expression in liver cancer correlates with suppres-sion of the hepatic phenotype and gain of metastatic properties. Oncogene28:3526 –3536.

27. Fu J, Xu D, Liu Z, Shi M, Zhao P, Fu B, Zhang Z, Yang H, Zhang H,Zhou C, Yao J, Jin L, Wang H, Yang Y, Fu YX, Wang FS. 2007.Increased regulatory T cells correlate with CD8 T-cell impairment andpoor survival in hepatocellular carcinoma patients. Gastroenterology 132:2328 –2339.

28. Wang S, Qiu L, Liu G, Li Y, Zhang X, Jin W, Gao GF, Kong X, MengS. 2011. Heat shock protein gp96 enhances humoral and T cell responses,decreases Treg frequency and potentiates the anti-HBV activity in BALB/cand transgenic mice. Vaccine 29:6342– 6351.

29. Li C, Cao S, Liu Z, Ye X, Chen L, Meng S. 2010. RNAi-mediateddownregulation of uPAR synergizes with targeting of HER2 through theERK pathway in breast cancer cells. Int. J. Cancer 127:1507–1516.

30. Kelly EJ, Hadac EM, Cullen BR, Russell SJ. 2010. MicroRNA antago-nism of the picornaviral life cycle: alternative mechanisms of interference.PLoS Pathog. 6:e1000820. doi:10.1371/journal.ppat.1000820.

31. Ebert MS, Sharp PA. 2010. MicroRNA sponges: progress and possibili-ties. RNA 16:2043–2050.

32. Jeong JH, Kwak DS, Rho HM, Jung G. 1996. The catalytic properties ofhuman hepatitis B virus polymerase. Biochem. Biophys. Res. Commun.223:264 –271.

33. Chien W, Pei L. 2000. A novel binding factor facilitates PTTG1 translo-cation and transcriptional activation. J. Biol. Chem. 275:19422–19427.

34. Smith VE, Franklyn JA, McCabe CJ. 2010. Pituitary tumor-transforminggene and its binding factor in endocrine cancer. Expert Rev. Mol. Med.12:e38.

35. Tong Y, Eigler T. 2009. Transcriptional targets for pituitary tumor-transforming gene-1. J. Mol. Endocrinol. 43:179 –185.

36. Chen Y, Shen A, Rider PJ, Yu Y, Wu K, Mu Y, Hao Q, Liu Y, Gong H,Zhu Y, Liu F, Wu J. 2011. A liver-specific microRNA binds to a highlyconserved RNA sequence of hepatitis B virus and negatively regulates viralgene expression and replication. FASEB J. 25:4511– 4521.

37. Fan CG, Wang CM, Tian C, Wang Y, Li L, Sun WS, Li RF, Liu YG.2011. miR-122 inhibits viral replication and cell proliferation in hepatitisB virus-related hepatocellular carcinoma and targets NDRG3. Oncol. Rep.26:1281–1286.

38. Ma F, Xu S, Liu X, Zhang Q, Xu X, Liu M, Hua M, Li N, Yao H, CaoX. 2011. The microRNA miR-29 controls innate and adaptive immuneresponses to intracellular bacterial infection by targeting interferon-.Nat. Immunol. 12:861– 869.

39. Sarasin-Filipowicz M, Krol J, Markiewicz I, Heim MH, Filipowicz W.2009. Decreased levels of microRNA miR-122 in individuals with hepatitisC responding poorly to interferon therapy. Nat. Med. 15:31–33.

40. Cermelli S, Ruggieri A, Marrero JA, Ioannou GN, Beretta L. 2011.Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease. PLoS One 6:e23937. doi:10.1371/journal.pone.0023937.

41. Zhang X, Zhang E, Ma Z, Pei R, Jiang M, Schlaak JF, Roggendorf M, LuM. 2011. Modulation of hepatitis B virus replication and hepatocyte dif-ferentiation by microRNA-1. Hepatology 53:1476 –1485.

42. Stiffler JD, Nguyen M, Sohn JA, Liu C, Kaplan D, Seeger C. 2009. Focaldistribution of hepatitis C virus RNA in infected livers. PLoS One 4:e6661.doi:10.1371/journal.pone.0006661.

43. Filipowicz W, Grosshans H. 2011. The liver-specific microRNA miR-122: biology and therapeutic potential. Prog. Drug Res. 67:221–238.

44. Quasdorff M, Protzer U. 2010. Control of hepatitis B virus at the level oftranscription. J. Viral Hepat. 17:527–536.

45. Smith VE, Franklyn JA, McCabe CJ. 2011. Expression and function ofthe novel proto-oncogene PBF in thyroid cancer–a new target for aug-menting radioiodine uptake. J. Endocrinol. 210:157–163.

46. Watkins RJ, Read ML, Smith VE, Sharma N, Reynolds GM, Buckley L,Doig C, Campbell MJ, Lewy G, Eggo MC, Loubiere LS, Franklyn JA,

Li et al.

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s://j

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asm

.org

/jour

nal/j

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embe

r 20

21 b

y 77

.38.

223.

73.

Boelaert K, McCabe CJ. 2010. Pituitary tumor transforming gene bindingfactor: a new gene in breast cancer. Cancer Res. 70:3739 –3749.

47. Molina-Jiménez F, Benedicto I, Murata M, Martín-Vílchez S, Seki T,Antonio Pintor-Toro J, Tortolero M, Moreno-Otero R, Okazaki K,Koike K, Barbero JL, Matsuzaki K, Majano PL, López-Cabrera M. 2010.Expression of pituitary tumor-transforming gene 1 (PTTG1)/securin inhepatitis B virus (HBV)-associated liver diseases: evidence for an HBV X

protein-mediated inhibition of PTTG1 ubiquitination and degradation.Hepatology 51:777–787.

48. Mizuguchi Y, Mishima T, Yokomuro S, Arima Y, Kawahigashi Y,Shigehara K, Kanda T, Yoshida H, Uchida E, Tajiri T, Takizawa T.2011. Sequencing and bioinformatics-based analyses of the microRNAtranscriptome in hepatitis B-related hepatocellular carcinoma. PLoS One6:e15304. doi:10.1371/journal.pone.0015304.

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