Hepatitis B virus (HBV) causes chronic infection in about 350 million people worldwide. Given the important role of the most abundant liver-specific microRNA, miR-122, in hepatic function and liver pathology, here we investigated the potential role and mechanism of miR-122 in regulating HBV replication. We found that miR-122 expression in liver was significantly down-regulated in patients with HBV infection compared with healthy controls, and the miR-122 levels were negatively correlated with intrahepatic viral load and hepatic necroinflammation. The depletion of endogenous miR-122 by its antisense inhibitor led to enhanced HBV replication, whereas overexpression of miR-122 by transfection of mimic or its expression vector inhibited viral production. We next identified cyclin G 1 as an miR-122 target from multiple candidate target genes that are involved in the regulation of HBV replication. Overexpression and knockdown studies both showed that cyclin G 1 regulated viral replication in HBV transfected cells. We also observed that cyclin G 1 expression was up-regulated in HBV-infected patients, and cyclin G 1 levels were inversely associated with miR-122 expression in liver tissues. Using coimmunoprecipitation, a luciferase reporter system, and electrophoretic mobility shift assay, we further demonstrated that cyclin G 1 specifically interacted with p53, and this interaction blocked the specific binding of p53 to HBV enhancer elements and simultaneously abrogated p53-mediated inhibition of HBV transcription. Finally, we show that miR-122 suppressed HBV replication in p53 wildtype cells but not in null isogenic cells. Conclusion: miR-122 down-regulates its target cyclin G 1 , and thus interrupts the interaction between cyclin G 1 and p53 and abrogates p53-mediated inhibition of HBV replication. Our work shows that miR-122 down-regulation induced by HBV infection can impact HBV replication and possibly contribute to viral persistence and carcinogenesis. (HEPATOLOGY 2012;55:730-741)

M icroRNAs (miRNAs) are a large family of small (ϳ21-nucleotide [nt]) noncoding RNAs that interact with complementary target sites in their target mRNAs to induce translational repression, deadenylation, and degradation (1). However, the reciprocal effect of target mRNA on miRNA activity is largely unknown. is the most abundant liverspecific miRNA, accounting for approximately 70% of the total miRNA population in the adult liver (2). It has been found to play key roles in liver development and hepatic function (3, 4), hepatocyte 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 (ϳ3.2 kb), enveloped, partially doublestranded DNA virus. The HBV genome contains four overlapping open reading frames (ORFs). The RNA transcripts are polyadenylated; capped; 3.5, 2.4, 2.1, and 0.7 kb in length; and named the pre-C/C or pregenomic RNA (pgRNA), pre-S, S, and X mRNAs, respectively. These mRNAs encode several overlapping viral proteins, including the polymerase, core, HBe, pre-S1, S2, S, and X proteins (14). There are approximately 350 million chronic HBV carriers worldwide, and chronic HBV infection is the major etiological factor in hepatocellular carcinoma (HCC) (15, 16). The relative risk for the development of HCC in chronic hepatitis B (CHB) patients is estimated to be 25 to 100 times higher than that in those without infection (15,17,18).Several possible pathways and molecular mechanisms have been reported for the involvement of HBV infection in malignant transformation of liver cells, including both direct and indirect mechanisms that likely act synergistically. Direct effects by viral factors include HBV DNA integration into the hepatocyte genome (which acts via cis-or trans-activation of nearby genes or enhances host chromosomal instability), the antiapoptotic and procarcinogenic functions of the HBx and truncated pre-S2/S viral proteins, and HBV mutants and genotypes (14,15,19,20). The indirect effects of chronic viral infection on malignant transformation include persistent inflammation and liver cirrhosis (which may significantly contribute to the transformation of hepatocytes and promote hepatocarcinogenesis through an integrated multistep process [21,22]), aberrant DNA methylation of specific cellular genes (23), and host susceptibility (24). However, the molecular mechanisms underlying HBV-induced carcinogenesis remain elusive and await further investigation (14, 15).Our previous study showed that loss of miR-122 induced by HBV infection enhances HBV replication through cyclin G1-modulated p53 activity, thereby possibly contributing to viral persistence (25). Moreover, miR-122 repression is only found in HCC arising in HBV-infected livers but not in HCV-infected liv-

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