| Background: HBV infection is the critical cause of acute and chronic hepatitis, which progresses to cirrhosis and hepatocellular carcinoma(HCC). More than 1,000,000 people die of HBV-related end-stage diseases each year, HBV infection has become the major etiological factor of threatening human health. So, the work of prevention HBV infection is very significanct. At present, HBV vaccine has been widely used, interferon alpha and nucleoside analogues have been used for the treatment of hepatitis B in clinic. However, there are respective defects which are vaccination failure, serious side effect and drug resistance. Therefore, it has important meaning to study the regulatory mechanism of HBV replication and the role of critical host factors in HBV infection, finally, confirm novel targets to therapy of HBV infection. SIRT1 is a NAD+-dependent class III histone deacetylase that is a structure component of the HBV cccDNA minichromosome, and it is closely related to a number of cellular functions. Increasing reports showed SIRT1 could regulate the process of virus infection. Whereas, whether the SIRT1 could directly regulate the life cycle of HBV is not very clear.Objective:1. To study the effect of SIRT1 on HBV replication and transcription.2. To elucidate the molecular mechanism of SIRT1 regulating HBV infection.3. To investigate the effect of SIRT1 inhibitor on HBV replication in vitro and in vivo.Methods:1. The mRNA and protein levels of SIRT1 in HBV stable replication cell lines(HepG2.2.15 and HepG2-HBV1.1 cells) and control cells(PHH and HepG2 cells) were detected by qRT-PCR and western blot, respectively. Furthermore, the effect of HBV transient expression on the mRNA and protein expression levels of SIRT1 was also detected by qRT-PCR and western blot, respectively.2. The HepG2.2.15 and Hep G2-HBV1.1 cells were transfected with lentiviruses containing sh RNAs targeting SIRT1(shCont, shSIRT1-1, shSIRT1-2) or plasmid expressing SIRT1. The effect of SIRT1 knockdown or SIRT1 overexpression on the expression of HBV DNA replicative intermediates was determined by real-time PCR and southern blot. Moreover, qRT-PCR and western blot were used to detect the effect of SIRT1 knockdown or SIRT1 overexpression on the expression of HBV 3.5kb mRNA and HBc, respectively. Finally, the role of knockdown SIRT1 or overexpression SIRT1 in the secretion of HBsAg and HBeAg was analyzed by ELISA.3. The effect of SIRT1 knockdown on HBV replication in Huh-7 cells transfected with pGEM-HBV1.3 or closed circular HBV genome was detected by real-time PCR and southern blot.4. The HepG2 cells were co-transfected with plasmids expressing HBV promoters or SIRT1, the activity of HBV four promoters was analyzed by dual-luciferase reporter system. Moreover, the activity of core promoter was furtherly analyzed in SIRT1-silencing HepG2 cells.5. QRT-PCR was used for analysis of the mRNA expression of various transcription factors related to HBV replication in HepG2.2.15 cells expressing sh Cont, shSIRT1-1 or shSIRT1-2. The effect of SIRT1 on the protein level of target transcription factor c-Jun, which is the subunit of AP-1, was measured by western blot.6. ChIP assays were performed to examine the interaction between c-Jun and HBV core promoter region.7. The binding site of c-Jun in the HBV core promoter region of pGEM-HBV1.3 was mutated. The effect of SIRT1 on HBV replication of the mutant pGEM-HBV1.3 was analyzed by real-time PCR and southern blot.8. Dual-luciferase assay was applied for analysing the effect of c-Jun knockdown on the enhancement of HBV core promoter activity induced by SIRT1 in HepG2 cells. In addition, real-time PCR and southern blot were used to detect the effect of c-Jun silencing on HBV replication in HepG2.2.15 and HepG2-HBV1.1 cells overexpressing SIRT1.9. The CC50 values of nicotinamide in HepAD38 and HepG2.2.15 cells were assayed by MTS.10. Hep AD38 and HepG2.2.15 cells were treated with various concentrations of nicotinamide. The effect of nicotinamide on HBV DNA replicative intermediates levels was detected by real-time PCR and southern blot. Moreover, qRT-PCR was applied for analyze the effect of nicotinamide on the transcription of HBV 3.5kb mRNA. Finally, the role of nicotinamide in the expression of HBc and the secretion of HBsAg and HBeAg was determined by western blot and ELISA, respectively.11. HBV-transgenic mice(C57BL/6J) were injected with various doses of nicotinamide solutions. The alanine transaminase(AST) and aspartate transaminase(ALT) levels in mice serum were determined by microplate method; the HBV DNA levels in mice serum and liver tissues were analyzed by real-time PCR; the HBsAg and HBeAg levels in mice serum were detected by ELISA.Results:1. Both mRNA and protein levels of SIRT1 were upregulated in HBV stable expressing cell lines relative to control cells. In addition, the mRNA and protein levels were also increased induced by HBV transient expression.2. Downregulation of SIRT1 in HepG2.2.15 and HepG2-HBV1.1 cells resulted in decreased levels of HBV DNA replicative intermediates(P<0.01), HBV3.5kb mRNA(P<0.01) and HBc. SIRT1 knockdown also inhibited HBs Ag and HBeAg secretion(P<0.05). Overexpression of SIRT1 increased the level of HBV DNA replicative intermediates(P<0.01) and HBV3.5kb mRNA(P<0.01), Furthermore, the overexpression of SIRT1 also resulted in the upregulation of HBsAg and HBeAg secretion(P<0.05).3. Gene silencing of SIRT1 also significantly inhibited the HBV DNA level in Huh-7 cells transfected with pGEM-HBV1.3 or closed circular HBV genome(P<0.05).4. Overexpression of SIRT1 significantly enhanced core promoter activity(P<0.001) but had no effect on SpI, SpII, and X promoter activities; In contrast, downregulation of SIRT1 suppressed nearly 50% of the core promoter activity(P<0.001).5. The mRNA(P<0.01) and protein levels of c-Jun subunit of AP-1 were decreased in SIRT1-silencing HepG2.2.15 cells. Overexpression of SIRT1 significantly increased c-Jun protein level.6. AP-1 could bind to HBV core promoter region.7. The enhancement of HBV replication induced by SIRT1 was abolished when the binding site of AP-1 in the HBV core promoter of pGEM-HBV1.3 was mutated.8. The HBV core promoter activity mediated by SIRT1 was abolished when c-Jun was suppressed. Moreover, silencing of c-Jun subunit of AP-1 also abolished enhancement of HBV replication induced by SIRT1.9. The CC50 values of nicotinamide in HepAD38 and HepG2.2.15 cells were 36.38 mM and 44.44 mM, respectively.10. Nicotinaminde treatment inhibited the expression of HBV DNA replicative intermediates(P<0.05), HBV3.5kb mRNA(P<0.01) and HBc in HepAD38 and HepG2.2.15. In addition, nicotinamide also reduced the secretion of HBs Ag and HBeAg(P<0.05).11. Nicotinaminde treatment had no effect on the levels of AST and ALT in mice serum. Nicotinamide decreased the level of serum HBV DNA in a dose dependent manner(P<0.05) as well as repressed HBs Ag and HBeAg level of the mice serum(P<0.05). More importantly, Lower levels of HBV DNA were found in the liver tissues of nicotinamide-treated mice than in the control mice(P<0.05).Conclusion:In this study, we investigated the role of SIRT1 in HBV replication. Our data showed that SIRT1 was significantly upregulated in HBV-expressing cell lines and that the silencing or overexpression of SIRT1 regulated HBV replication. Further, SIRT1 enhanced HBV core promoter activity through transcription factor AP-1. Finally, SIRT1 inhibitor nicotinamined also markedly suppressed HBV DNA replication in vitro and in vivo. Our data suggested a role for SIRT1 in HBV replication and identified it as a novel host factor in the HBV replication process. |
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