Lizhao Chen

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-induced down-regulation of HO-1 negatively affects miR-122-mediated suppression of HBV

As the most abundant liver-specific microRNA (miRNA), miR-122 has been extensively studied for its role in the regulation of lipid metabolism, hepatocarcinogenesis and hepatitis C virus (HCV) replication, but little is known regarding its role in the replication of Hepatitis B virus (HBV), a highly prevalent hepatotropic virus that can cause life-threatening complications. In this study we examined the effects of antisense inhibition of miR-122 and transfection of a miR-122 mimic on HBV expression in hepatoma cells. The over-expression of miR-122 inhibited HBV expression, whereas the depletion of endogenous miR-122 resulted in increased production of HBV in transfected cells. We further found that the down-regulation of Heme oxygenase-1 (HO-1) by miR-122 plays a negative role in the miR-122-mediated inhibition of viral expression. Our study demonstrates the anti-HBV activity of miR-122, suggesting that therapies that increase miR-122 and HO-1 may be an effective strategy to limit HBV replication.

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.

Treg suppress CTL responses upon immunization with HSP gp96

HSP gp96‐based vaccines have been trialled in rodent models and, more recently, in humans. Better understanding of gp96s immunomodulatory role will help with the design of more effective strategies for treatment of cancer and infectious diseases. In this study, we monitored the activities of T cells and activation of Treg in BABL/c mice after immunization using different doses of gp96 as adjuvant. We found that co‐injection of gp96 simultaneously stimulated both CTL and Treg activity. Activation of CTL at low dose was far more pronounced than Treg activation. Treg population and suppression increased with gp96 dose, eventually abrogating the T‐cell response induced by immunization. Low‐dose cyclophosphamide treatment could restore the T‐cell responses lost after high‐dose gp96 adjuvant injection by suppression of Treg activation. We further examined the effect of different doses of gp96 or N355 peptide administration on tumor rejection. Our results provide new insights into the mechanisms of gp96‐mediated balance between regulatory and responder T cells, which may facilitate future development of an effective gp96‐based therapeutic vaccine.

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.

RNAi-mediated downregulation of uPAR synergizes with targeting of HER2 through the ERK pathway in breast cancer cells

Overexpression of urokinase plasminogen activator receptor (uPAR) or HER2 (erbB‐2) in breast cancer is associated with a poor prognosis. We previously reported that gene amplification and overexpression of HER2 and uPAR occur in 70% of HER2‐amplified tumor cells from blood or tissue of patients with breast cancer. In this study, we first examined whether depletion of HER2 and uPAR synergized in suppression of the growth of breast cancer cells that overexpress both HER2 and uPAR (SKBR3 and ZR 751). The results showed that depletion of either HER2 or uPAR by RNA interference suppressed cell growth and induced cell apoptosis, but that these effects were significantly enhanced in cells depleted of both HER2 and uPAR. Mechanistic analysis demonstrated that silencing of HER2 and uPAR caused suppression of MAPK signal pathways, resulting in decrease of ERK activity and prompting a high p38/ERK activity ratio. The level of the phosphorylated form of ERK was decreased in cells depleted of HER2, uPAR or both, and the effect in cells depleted of both is the most evident. Moreover, downregulation of uPAR synergized with trastuzumab to suppress the growth and induce apoptosis of SKBR3 and ZR 751 cells. uPAR RNAi significantly enhanced the effect of trastuzumab on inhibition of MAPK signal pathways. In conclusion, targeting HER2 and uPAR has a synergistic inhibitory effect on breast cancer cells. Our results provide evidence that simultaneous downregulation of HER2 and uPAR may offer an effective tool for breast cancer therapy.

Chronic inflammation contributes to the development of hepatocellular carcinoma by decreasing miR-122 levels

Persistent inflammation in chronic hepatitis plays a major role in the development of hepatocellular carcinoma (HCC). In this study, the major inflammatory cytokines expressed in chronic hepatitis, IL-6 and TNF-α, induced a marked decrease in microRNA-122 (miR-122) levels, and miR-122 expression was downregulated in the livers of chronic hepatitis B (CHB) patients. The decrease of miR-122 caused upregulation of the proinflammatory chemokine CCL2. IL-6 and TNF-α suppressed miR-122 both by directly downregulating the transcription factor C/EBPα and indirectly upregulating c-myc, which blocks C/EBPα-mediated miR-122 transcription. In addition, IL-6 and TNF-α levels were elevated and miR-122 levels were decreased in mouse and rat models of diethylnitrosamine (DEN)-induced HCC. Restoration of miR-122 levels through delivery of agomir-122 suppressed DEN-induced hepatocarcinogenesis in mice. Our results show that inflammation-induced miR-122 downregulation in hepatitis contributes to carcinogenesis and suggest that increasing miR-122 may be an effective strategy for preventing HCC development in CHB patients.

TAT-mediated gp96 transduction to APCs enhances gp96-induced antiviral and antitumor T cell responses.

The heat shock protein gp96 is an adjuvant that can elicit T cell responses against cancer and infectious diseases, via antigen presentation, in both rodent models and clinical trials. Its uptake and internalization into antigen presenting cells (APCs) is a critical step in gp96-mediated immune responses. This study examined strategies to improve the cell internalization and T cell activation of gp96. It was found that recombinant fusion with the cell-penetrating peptide TAT (trans-activator of transcription) slightly decreased the aggregation level of gp96 and significantly increased its internalization into macrophages. Furthermore, immunization with the TAT-gp96 fusion dramatically enhanced gp96-mediated hepatitis B virus (HBV)-specific T cell responses and its antiviral efficiency in HBV transgenic mice compared to rgp96. In addition, the inclusion of TAT significantly improved the antitumor T cell immune response to a gp96 vaccine in the B16 melanoma model. These results provide evidence that the efficient transduction of gp96 into APCs can significantly enhance the outcome of gp96-based immunotherapy, and therefore provide a basis for more efficient approaches to improving the immunoregulatory and adjuvant functions of this unique T cell adjuvant.

Placenta-derived gp96 as a multivalent prophylactic cancer vaccine

A major challenge for designing prophylactic cancer vaccines is to define immunogenic and safe cancer antigens. Given the striking similarity of antigen expression patterns between cancer and embryonic tissues, we defined a prototype strategy of using placenta-derived heat shock protein gp96, which induces prophylactic anti-tumor T cell responses. Immunization with placental gp96 provided partial protection and long-term (at least 3 months) anti-tumor immunity against growth of transplantable melanoma or breast tumors in mice, elicited total protection against 7, 12-dimethylbenz(a)-anthracene (DMBA)-induced mammary tumors in rats, and significantly reduced the occurrence and growth of autochthonous breast tumors in HER2 transgenic mice. Placental gp96 activated HER2- and MUC1-specific T cell responses through binding to tumor-associated antigens. Our results reveal the novel immunogenicity of placental gp96 and its potential use as a multivalent cancer vaccine.