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-

ABSTRACT

More than 350 million people are chronically infected with hepatitis B virus, and dysfunctional T cell responses contribute to persistent viral infection and immunopathogenesis in chronic hepatitis B (CHB). However, the underlying mechanisms of T cell hyporesponsiveness remain largely undefined. Given the important role of microRNA-146a (miR-146a) in diverse aspects of lymphocyte function, we investigated the potential role and mechanism of miR-146a in regulating T cell immune responses in CHB. We found that miR-146a expression in T cells is significantly upregulated in CHB compared with healthy controls, and miR-146a levels were correlated with serum alanine aminotransaminase levels. Both inflammatory cytokines and viral factors led to miR-146a upregulation in T cells. Stat1 was identified as a miR-146a target that is involved in antiviral cytokine production and the cytotoxicity of CD4+ and CD8+ T cells. In vitro blockage of miR-146a in T cells in CHB greatly enhanced virus-specific T cell activity. Therefore, our work demonstrates that miR-146a upregulation in CHB causes impaired T cell function, which may contribute to immune defects and immunopathogenesis during chronic viral infection.

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A round 400 million people worldwide are infected with hepatitis B virus (HBV). Chronic hepatitis B (CHB), which is triggered by HBV infection, results in a huge health burden on the global community, as it is correlated with a significantly increased risk for the development of cirrhosis, liver failure, and hepatocellular carcinoma (HCC) (1). Currently, treatment of CHB consists mainly of pegylated alpha interferon (IFN-␣) and nucleoside or nucleotide analogs (e.g., lamivudine, adefovir, and entecavir). IFN-␣ was the first drug licensed to treat HBV infection. As an important first-line treatment option, pegylated IFN-␣ as monotherapy can effectively treat CHB in 25 to 40% of patients, and greater sustained virological responses (SVRs) and hepatitis B virus e antigen (HBeAg) seroconversion rates in HBeAg-positive patients were observed with addition of nucleoside/nucleotide analogue therapies (2, 3). In fact, treatment with pegylated IFN results in the highest rate of off-treatment sustained responses among currently available drugs (4). Moreover, responses to IFNbased therapy are accompanied by the potential for hepatitis B virus surface antigen (HBsAg) loss or seroconversion, and early serum HBsAg loss was recently reported to have predictive value for SVRs to IFN in both HBeAg-positive and -negative CHB patients (5-7).As a member of the type I interferons, IFN-␣ can initiate the activation of Jak/STAT and NF-B signaling, which induces hundreds of IFN-stimulated genes (ISGs) and may play an important role in IFN-mediated anti-HBV activity. Both in vitro and in vivo studies have shown that besides a stimulating effect on cytotoxic T lymphocytes and natural killer cell function, IFN-based therapy (IFN-␣-2b and pegylated IFN-␣-2a or -2b) also has a direct antiviral effect by preventing the formation or accelerating decay of viral capsids and/or inducing antiviral ISGs that inhibit HBV expression and replication (8-13). Inhibition of IFN-␣ signaling by HBV has been suggested to antagonize the IFN response (14).Nevertheless, these studies also strongly suggest that there is significant potential, in principle, to modulate the effectiveness of IFN-mediated anti-HBV activities. Moreover, the antiviral activity of ISGs remains elusive and still awaits further investigation (15). Responses to IFN-␣ therapy vary greatly in CHB patients, but the underlying mechanisms are almost unknown (4-6). Notably, IFN-␣/␤ was recently found to suppress HBV replication in HBV transgenic mice when the viral load was high, whereas it enhanced HBV replication when the viral load was low, indicating its dual function for HBV (16). Taken together, the data show that the precise mechanism of action of IFN-␣ has not been understood fully.MicroRNAs (miRNAs) are a class of small RNAs of approximately 22 nucleotides (nt) which interact with complementary target sites, usually in the 3=-untranslated region (3=-UTR) of target mRNAs, and induce their translational repression, deadenylation, and degradation. MicroRNA-122 (miR-122), a mammalian liv...

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