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Changfei Li,
Yanzhong Wang, Saifeng Wang,
Bo Wu,
Junli Hao,
Hongxia Fan,
Ying Ju,
Yuping Ding,
Lizhao Chen,
Xiaoyu Chu,
Wenjun Liu,
Xin Ye,
Songdong Meng
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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. In this study, we provide evidence that all HBV mRNAs harboring a 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 (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 up-regulated in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC). Over-expression and knock-down 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 mRNAs-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.
Journal of Virology 12/2012; · 5.40 Impact Factor
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Junli Hao,
Wensong Jin,
Xinghui Li, Saifeng Wang,
Xiaojun Zhang,
Hongxia Fan,
Changfei Li,
Lizhao Chen,
Bin Gao,
Guangze Liu,
Songdong Meng
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ABSTRACT: 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 marked decrease of miR-122 expression in hepatocytes. We next showed that IFN-α-induced miR-122 down-regulation 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' -UTR, indicating that 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 up-regulation of NT5C3 mRNA expression by 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 significantly enhanced when blocking IFN-α-induced down-regulation of miR-122. We therefore conclude that IFN-α-induced inhibition of miR-122 may negatively affect the anti-HBV function of IFN-α. These data provide valuable insight for better understanding of the anti-viral mechanism of IFN-α and raise further potential interest in enhancing its anti-HBV efficacy.
Journal of Virology 10/2012; · 5.40 Impact Factor
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ABSTRACT: Heat-shock protein gp96 associates with antigenic peptides derived from tumor and virus. Exogenous gp96-peptide complexes are taken up by antigen-presenting cells through interaction with its receptor CD91 on the cell surface, and cross-present antigenic peptides to MHC class I molecules by a peptide relay line in the endoplasmic reticulum for specific T-cell activation. Meanwhile, gp96 has been shown to initiate innate immune responses through interaction with toll-like receptor 2 and toll-like receptor 4. Recent studies have shown a gp96-mediated immune balance between CTL and Tregs. With the further understanding of counteracting immunosuppressive mechanisms in gp96-induced cellular immune responses, and establishment of high level production of recombinant gp96 by the yeast, gp96 appears to be a promising candidate for designing effective therapeutic vaccines against tumor and infectious diseases.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 03/2012; 28(3):261-6.
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Saifeng Wang,
Lipeng Qiu,
Xiaoli Yan,
Wensong Jin,
Yanzhong Wang,
Lizhao Chen,
Erjie Wu,
Xin Ye,
George F Gao,
Fusheng Wang,
Yu Chen,
Zhongping Duan,
Songdong Meng
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ABSTRACT: 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 11/2011; 55(3):730-41. · 11.66 Impact Factor
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Xiaoli Yan,
Xiaojun Zhang,
Yanzhong Wang,
Xinghui Li, Saifeng Wang,
Bao Zhao,
Yang Li,
Ying Ju,
Lizhao Chen,
Wenjun Liu,
Songdong Meng
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ABSTRACT: Despite its potent immunostimulatory properties, vaccination with autologous tumor-derived gp96 has relatively modest antitumor effect in a range of clinical trials. Based on our previous study showing a gp96-mediated immune balance between CTL and Tregs, here we investigated possible synergy between gp96 vaccine and systemic Treg depletion on induction of antitumor T-cell immunity and the mechanisms accounting for synergistic efficacy. In gp96-peptide complex immunized BALB/c mice, anti-CD25 mAb treatment significantly increased IFN-γ-producing CD8(+) and CD4(+) T cells by about 1-2-fold in spleen and 40-50% in lymph node. A significantly higher number of peptide-specific CTL were observed under anti-CD25 mAb treatment compared with no treatment. Moreover, Treg depletion synergistically improved the anticancer activity of tumor-derived gp96 vaccine in the poorly immunogenic and highly tumorigenic B16 melanoma model in C57BL/6 J mice. While gp96 immunization alone led to the modest enhancement of CTL activities in spleen, the combination with Treg depletion dramatically increased tumor-specific CTL responses. In addition, the combination resulted in a significant increase of CD8(+) T-cell infiltration in tumor, which correlated with an enhanced inhibition of tumor growth. Our results provide evidence that targeting Tregs may provide a more efficient strategy to potentiate gp96-mediated T-cell responses and enhance the antitumor efficiency of gp96-based therapeutic vaccine.
Cancer Immunology and Immunotherapy 07/2011; 60(12):1763-74. · 3.70 Impact Factor
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ABSTRACT: 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.
Vaccine 05/2011; 29(37):6342-51. · 3.77 Impact Factor
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ABSTRACT: While currently therapeutic vaccines for chronic hepatitis B virus (HBV) infection are actively being developed to complement standard antiviral treatments, their immune activity, especially T cell activity, remains to be further improved. Here, we investigated the role of heat shock proteins HSP70 and gp96 on cellular and humoral immunity, using the main structure antigens of hepatitis core (HBcAg) and surface (HBsAg) as the DNA vaccine. By ELISPOT (enzyme linked immunospot assay), IFN-gamma intracellular staining, [3H]-thymidine incorporation and ELISA (enzyme linked immunosorbent assay) analyses, we showed that immunization with HBsAg/HBcAg DNA formulation along with HSP70 or gp96 induced significant increase of T-cell (about 1-6-fold) and antibody (about 20%-60%) immunity against HBsAg and HBcAg. These results may provide bases for designing HSP70- and gp96-based vaccines aimed at eliciting T-cell responses for therapeutic applications.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 05/2011; 27(5):790-8.
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Yang Li,
Haolei Song,
Jin Li,
Yanzhong Wang,
Xiaoli Yan,
Bao Zhao,
Xiaojun Zhang, Saifeng Wang,
Lizhao Chen,
Bingsheng Qiu,
Songdong Meng
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ABSTRACT: 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.
Journal of biotechnology 02/2011; 151(4):343-9. · 2.88 Impact Factor
