Saifeng Wang

Loss of microRNA 122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity†‡

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 G1 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 G1 regulated viral replication in HBV transfected cells. We also observed that cyclin G1 expression was up‐regulated in HBV‐infected patients, and cyclin G1 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 G1 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 G1, and thus interrupts the interaction between cyclin G1 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;)

MiR-122 in hepatic function and liver diseases

As the most abundant liver-specific microRNA, microRNA-122 (miR-122) is involved in various physiological processes in hepatic function as well as in liver pathology. There is now compelling evidence that miR-122, as a regulator of gene networks and pathways in hepatocytes, plays a central role in diverse aspects of hepatic function and in the progress of liver diseases. This liver-enriched transcription factors-regulated miRNA promotes differentiation of hepatocytes and regulates lipid metabolism. With regard to liver diseases, miR-122 was shown to stimulate hepatitis C virus (HCV) replication through a unique and unusual interaction with two binding sites in the 5′-UTR of HCV genome to mediate the stability of the viral RNA, whereas inhibit the expression and replication of hepatitis B virus (HBV) by a miR-122-cylin G1/p53-HBV enhancer regulatory pathway. In addition, miR-122 acts as a suppressor of cell proliferation and malignant transformation of hepatocytes with remarkable tumor inhibition activity. Notably, a clinical trial targeting miR-122 with the anti-miR-122 oligonucleotides miravirsen, the first miRNA targeted drug, has been initiated for treatment of HCV infection. With further understanding of the comprehensive roles of miR-122 in hepatic functions and the mechanisms involved in miR-122 down-regulation in chronic hepatitis or hepatocellular carcinoma, miR-122 appears to be a promising candidate for effective therapeutic approaches against tumor and infectious diseases.

Hepatitis B Virus mRNA-Mediated miR-122 Inhibition Upregulates PTTG1-Binding Protein, Which Promotes Hepatocellular Carcinoma Tumor Growth and Cell Invasion

ABSTRACT As the most abundant liver-specific microRNA, miR-122 is involved in diverse aspects of hepatic function and neoplastic transformation. Our previous study showed that miR-122 levels are significantly decreased in hepatitis B virus (HBV)-infected patients, 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 with hepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity.). In this study, we provide evidence that all HBV mRNAs harboring an miR-122 complementary site act as sponges to bind and sequester endogenous miR-122, indicating that the highly redundant HBV transcripts are involved in HBV-mediated miR-122 suppression. We next identified pituitary tumor-transforming gene 1 (PTTG1) binding factor (PBF) as a target of miR-122 and demonstrated that HBV replication causes an obvious increase in PBF levels. Furthermore, we observed that the miR-122 levels were decreased and PBF was upregulated in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). Overexpression and knockdown studies both revealed that PBF enhances proliferation and invasion of HCC cells, and silencing PBF resulted in a dramatic reduction of HCC tumor growth 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 regulatory pathway that facilitates malignant hepatocyte growth and invasion in CHB which may contribute to CHB-induced HCC development and progression. Our work underscores the reciprocal interplay of host miRNA sequestration and depletion by viral mRNAs, which may contribute to chronic-infection-related cancer.

MicroRNA-146a Feedback Suppresses T Cell Immune Function by Targeting Stat1 in Patients with Chronic Hepatitis B

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.

Inhibition of Alpha Interferon (IFN-α)-Induced MicroRNA-122 Negatively Affects the Anti-Hepatitis B Virus Efficiency of IFN-α

ABSTRACT Alpha interferon (IFN-α)-based therapy can effectively treat chronic hepatitis B virus (HBV) infection, which causes life-threatening complications. Responses to IFN-α therapy vary greatly in chronic hepatitis B (CHB) patients, but underlying mechanisms are almost unknown. In this study, we found that IFN-α treatment induced a marked decrease of microRNA-122 (miR-122) expression in hepatocytes. We next showed that IFN-α-induced miR-122 downregulation was only partly due to transcriptional suppression. One IFN-stimulated gene (ISG), NT5C3, which was identified as a miR-122 target, efficiently inhibited miR-122 by binding and sequestering miR-122 with its mRNA 3′-untranslated region (3′-UTR), indicating that this ISG is involved in IFN-α-mediated miR-122 suppression. Notably, the inhibitory effect of IFN-α on miR-122 was completely abolished by blocking IFN-α-induced upregulation of NT5C3 mRNA expression by RNA interference (RNAi). Meanwhile, we observed that miR-122 dramatically inhibited HBV expression and replication. Finally, we showed that IFN-α-mediated HBV-inhibitory effects could be enhanced significantly by blocking IFN-α-induced downregulation of miR-122. We therefore concluded that IFN-α-induced inhibition of miR-122 may negatively affect the anti-HBV function of IFN-α. These data provide valuable insights for a better understanding of the antiviral mechanism of IFN-α and raise further potential interest in enhancing its anti-HBV efficacy.

Heat shock protein gp96 enhances humoral and T cell responses, decreases Treg frequency and potentiates the anti-HBV activity in BALB/c and transgenic mice

More than 350 million people worldwide are chronically infected with hepatitis B virus (HBV). Broad repertoire and strong magnitude of HBV-specific T cell responses are thought to play key roles for virus control and clearance. Previous studies together with ours showed that heat shock protein gp96 as adjuvant induces antigen specific T cell responses, yet little is known for its anti-viral properties. Here, we investigated the role of gp96 mediated cellular and humoral immunity in antiviral effects in HBV transgenic mice. Immunization with HBV surface (HBsAg) and core (HBcAg) antigens combined formulation along with gp96 induced robust antiviral T-cell and antibody immunity against HBsAg and HBcAg. Compared with non-immunized control, immunization with gp96 adjuvant vaccine led to decrease of serum HBs level and HBc expression in hepatocyte by 45% and 90% at maximum, respectively, and decreased serum HBV-DNA level to below or close to the detection limit 4 weeks after the last immunization, suggesting the therapeutic effect. A significant enhancement in cellular responses towards HBcAg and increased infiltration of CD8+ T cells in liver of transgenic were observed under treatment with gp96 compared with no treatment (P<0.05 or 0.01). Treatment with gp96 was capable of reducing Tregs by overall 30-40%. The superior immune responses induced with the aid of gp96 correlated with improved antiviral effect by vaccination with HBsAg and HBcAg. We conclude that gp96 may contribute to enhanced antiviral immunity in transgenic mice at least partly by Treg down-regulation. HBcAg may act as potent adjuvant for Th1 response. Our study reveals the novel property of gp96 in immune modulation and its potential use for breaking immunotolerance in immunotherapy of chronic HBV infection.

Hansenula polymorpha expressed heat shock protein gp96 exerts potent T cell activation activity as an adjuvant

Previous studies together with ours showed that heat shock protein gp96 as an adjuvant induces antigen specific T cell responses against cancer and infectious diseases. However, at present there is no efficient method to obtain high amount of full-length gp96 by in vitro expression. Here, we used the yeast Hansenula polymorpha as an efficient host for gp96 recombinant protein production. The transformant clones with highly expressed recombinant proteins were screened and selected by measuring the halo size which indicates enzymatic hydrolysis of starch in the medium. High-level production of gp96 (around 150mg/mL) was achieved by using high-cell density fed-batch cultivations. We showed that peptide binding of the recombinant protein has similar specificity and intrinsic binding parameters as that of the native gp96. We next examined the self-assembly properties and high-order structures of the recombinant protein. Moreover, the H. polymorpha expressed recombinant gp96 can effectively induce HBV-specific CTL response in immunized mice while Escherichia coli-expressed gp96 cannot. Our results therefore may provide bases for structure and functional studies of gp96 and thereby potentially expedite the development of gp96-based vaccines for immunotherapy of cancer or infectious diseases.