Changfei Li

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.

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.

Competitive virus and host RNAs: the interplay of a hidden virus and host interaction

During virus infection, viral RNAs and mRNAs function as blueprints for viral protein synthesis and possibly as pathogen-associated molecular patterns (PAMPs) in innate immunity. Here, considering recent research progress in microRNAs (miRNAs) and competitive endogenous RNAs (ceRNAs), we speculate that viral RNAs act as sponges and can sequester endogenous miRNAs within infected cells, thus cross-regulating the stability and translational efficiency of host mRNAs with shared miRNA response elements. This cross-talk and these reciprocal interactions between viral RNAs and host mRNAs are termed “competitive viral and host RNAs” (cvhRNAs). We further provide recent experimental evidence for the existence of cvhRNAs networks in hepatitis B virus (HBV), as well as Herpesvirus saimiri (HVS), lytic murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) infections. In addition, the cvhRNA hypothesis also predicts possible cross-regulation between host and other viruses, such as hepatitis C virus (HCV), HIV, influenza virus, human papillomaviruses (HPV). Since the interaction between miRNAs and viral RNAs also inevitably leads to repression of viral RNA function, we speculate that virus may evolve either to employ cvhRNA networks or to avoid miRNA targeting for optimal fitness within the host. CvhRNA networks may therefore play a fundamental role in the regulation of viral replication, infection establishment, and viral pathogenesis.

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.

Heat shock protein gp96 adjuvant induces T cell responses and cross-protection to a split influenza vaccine

The commonly used inactivated or split influenza vaccines induce only induce minimal T cell responses and are less effective in preventing heterologous virus infection. Thus, developing cross-protective influenza vaccines against the spread of a new influenza virus is an important strategy against pandemic emergence. Here we demonstrated that immunization with heat shock protein gp96 as adjuvant led to a dramatic increased antigen-specific T cell response to a pandemic H1N1 split vaccine. Notably, gp96 elicited a cross-protective CD8(+) T cell response to the internal conserved viral protein NP. Although the split pH1N1vaccine alone has low cross-protective efficiency, adding gp96 as an adjuvant effectively improved the cross-protection against challenge with a heterologous virus in mice. Our study reveals the novel property of gp96 in boosting the T cell response against conserved epitopes of influenza virus and its potential use as an adjuvant for human pre-pandemic inactivated influenza vaccines against different viral subtypes.

Heat shock protein gp96 decreases p53 stability by regulating Mdm2 E3 ligase activity in liver cancer

The resistance to apoptosis displayed by liver cancer plays a key role in hepatocarcinogenesis, tumor progression, and resistance to chemo- or radio-therapy. In this study, we uncovered the potential role and mechanism of heat shock protein gp96 in regulating liver tumor cell growth and apoptosis. P53 protein was identified as a gp96 client protein by profiling apoptosis-related proteins in gp96-knockdown liver cancer cells. Overexpression and knockdown studies both demonstrated that gp96 decreases p53 protein levels, and gp96 regulated cell apoptosis in a p53-dependent manner. We further provide evidence that gp96 interacts with both p53 and Mdm2 to enhance Mdm2-mediated p53 ubiquitination and degradation. Moreover, targeting gp96 with siRNA induced cell apoptosis and led to the suppression of liver tumor growth in vivo. In conclusion, we elucidated an underlying mechanism by which gp96 promotes p53 degradation via increasing Mdm2 E3 ligase activity and provided a new therapeutic strategy to target the gp96-mediated anti-apoptotic characteristic of hepatocellular carcinoma.