Effect Of SIRT6 Inhibitor,OSS₁28167 On HBV Replication

Background: Hepatitis B virus infection is a major public health problem.It is estimated that there are 257 million chronic HBV infections in the world,including 86 million in China.Chronic HBV infection,which is harmful to human health,can gradually develop into hepatitis,cirrhosis and liver cancer.Every year,about 650000 people die of various end-stage diseases caused by HBV infection.At present,the clinical drugs for treating chronic HBV infection are mainly nucleoside analogues and interferon,but neither of them can completely eliminate HBV,therefore,screening a new anti HBV drug is very important.By screening a library of3000 compounds,we found that SIRT6 inhibitor,OSS₁28167 could effectively inhibit HBV replication.The research used HBV stable expression cell line HepG2.2.15,HBV infection cell model HepG2-NTCP and HBV transgenic mouse to comprehensively analyze the effect of OSS₁28167 on HBV replication,and further elucidated the molecular mechanism of its antiviral effect.Objective:1.To study the effect of SIRT6 inhibitor OSS₁28167 on HBV replication in HepG2.2.15 and HepG2-NTCP cell lines.2.To study the antiviral effect of OSS₁28167 in HBV transgenic mouse.3.To elucidate the molecular mechanism of the antiviral effect of OSS₁28167.Methods:1.The toxicity of OSS₁28167 on HepG2.2.15 and HepG2-NTCP cells was analyzed by MTS.2.The HepG2.2.15 cells and HBV infected HepG2-NTCP cells were treated with 100μM OSS₁28167.Real-time PCR and southern blot were used to detect the effect of OSS₁28167 on HBV core DNA level.QRT-PCR was performed to determine the level of HBV 3.5-kb RNA in OSS₁28167-treating cells.The effect of OSS₁28167 on HBsAg and HBeAg secretions were assayed by ELISA.Western blot was applied for detecting the effect of OSS₁28167 on HBs protein expression.3.In HBV transgenic mice,50mg/kg OSS₁28167 solution was injected intraperitoneally.Firsty,the level of ALT in mice serum was analyzed by light colorimetry.Then,real-time PCR was performed to detect HBV DNA level in mice serum;ELISA was performed to detect HBsAg and HBeAg levels in mice serum.Further,the level of HBV DNA in mice tissue was examined by real-time PCR;The levels of total HBVRNAs and 3.5-kb RNA in mice tissue were examined by qRT-PCR;IHC was applied for detecting the protein level of HBc in mice tissue.4.ShSIRT6 plasmid was transfected into HepG2.2.15 cells and HBV infected HepG2-NTCP cells.Real-time PCR and southern blot were used to detect the HBV core DNA level in SIRT6 silencing cells.QRT-PCR was applied for analyzing the effect of SIRT6 silencing on total HBV RNAs and 3.5-kb RNA levels.Moreover,the effect of SIRT6 silencing on the expression of HBc and HBs protein were detected by IFC and western blot,respectively.The role of SIRT6 silencing in HBsAg and HBeAg secretions were analyzed by ELISA.Finally,the effect of SIRT6 silencing on HBV cccDNA level was analyzed by Taqman probe specific real-time PCR.5.HepG2.2.15 cells and HBV infected HepG2-NTCP cells were transfected with Flag-SIRT6 plasmid,at the same time,both cells were treated with 100μM OSS₁28167.Firstly,western blot was used to analyze the effect of OSS₁28167 on SIRT6 protein expression and deacetylase activity.Then,real-time PCR and southern blot were performed to detect HBV core DNA level;QRT-PCR was used to determine total HBV RNAs and 3.5-kb RNA levels;Taqman probe specific real-time PCR was used to examine HBV cccDNA level.Finally,the role of OSS₁28167 in promotion of HBV replication and transcription induced by SIRT6 were analyzed.6.Huh7 cells were co-transfected with HBV promoter and Flag-SIRT6 plasmid.The effect of SIRT6 overexpression on the activity of HBV promoter was analyzed by dual-luciferase assay.7.HepG2.2.15 cells were transfected with Flag-SIRT6 plasmid.QRT-PCR was used to screen the effect of SIRT6 on the mRNA levels of transcription factors related to HBV CP activity.Western blot was further applied for analyzing the effect of SIRT6 on the expression of PPARαprotein.8.ShSIRT6 plasmid was transfected into HepG2.2.15 cells.QRT-PCR and western blot were used to analyze the role of SIRT6 silencing in PPARα mRNA and protein expression,respectively.9.HepG2.2.15 cells were treated with 100μM OSS₁28167.The effect of OSS₁28167 on PPARα mRNA and protein expression were determined by qRT-PCR and western blot,respectively.10.Huh7 cells were co-transfected with pGL3-Cp and shPPARαplasmid,and the effect of silencing PPARα on HBV CP activity was verified by dual-luciferase assay.11.ShPPARα plasmid was transfected into HepG2.2.15 cells.Real-time PCR and qRT-PCR were performed to analyze the effect of silencing PPARα on the levels of HBV core DNA and HBV 3.5-kb RNA,respectively.The effect of silencing PPARα on HBsAg and HBeAg secretions were assayed by ELISA.Western blot was used to examine the effect of silencing PPARα on HBs protein expression.12.HepG2.2.15 cells and HBV infected HepG2-NTCP cells were cotransfected with Flag-SIRT6 and shPPARα plasmid.Real time PCR and southern blot were used to detect HBV core DNA level;QRT-PCR was performed to determine HBV 3.5-kb RNA level.Then the role of PPARαin enhancement of HBV replication and transcription regulated by SIRT6 were analyzed.13.HepG2.2.15 cells and HBV infected HepG2-NTCP cells were treated with 100μM OSS₁28167,then,both cells were transfected with Flag-SIRT6 or Flag-PPARα plasmid.Real time PCR and southern blot were used to detect HBV core DNA level;QRT-PCR was performed to determine HBV 3.5-kb RNA level.Then the effect of SIRT6 and PPARαon the inhibitory of HBV replication and transcription regulated by OSS₁28167 were analyzed.Results:1.OSS₁28167 had no cytotoxicity on both HepG2.2.15 and HepG2-NTCP cells within 400 μM.2.OSS₁28167 significantly decreased HBV core DNA level（P<0.05）.The level of HBV 3.5-kb RNA was also modestly decreased after treated with OSS₁28167（P<0.05）.Moreover,OSS₁28167 treatment inhibited HBsAg and HBeAg secretions,as well as HBs protein expression in cell lysates（P<0.05）.3.OSS₁28167 treatment had no effect on the levels of ALT in miceserum.Encouragingly,treatment with OSS₁28167 resulted in a significant reduction of HBV DNA in serum,as well as serum HBsAg and HBeAg in time-dependent manner（P<0.05）.Moreover,treatment with OSS₁28167resulted in a marked reduction of intrahepatic HBV DNA,total HBV RNAs and 3.5-kb RNA levels（P<0.05）.Lower level of HBc protein was found in the liver tissues of OSS₁28167-treated mice than in the control mice.4.In SIRT6 silencing group,the decreased levels of HBV core DNA,total HBV RNAs and 3.5-kb RNA were observed（P<0.05）.In addition,SIRT6 silencing moderately reduced HBsAg and HBeAg secretions,as well as HBs and HBc protein expression（P<0.05）.Interestingly,SIRT6 knockdown had no significant effect on HBV cccDNA level.5.SIRT6 deacetylase activity was dramatically decreased in OSS₁28167-treated cells,however,OSS₁28167 had no significant effect on SIRT6 protein expression（P<0.05）.Consistently,SIRT6 overexpression promoted HBV transcription and replication while abolished by OSS₁28167 treatment.6.SIRT6 overexpression significantly increased the activity of HBV core promoter（P<0.05）,whereas the others had no obvious alteration.7.The mRNA and protein levels of PPARα were markedly increased in SIRT6 ectopic expression cells（P<0.05）.8.In contrast,both mRNA and protein levels of PPARα weredecreased in SIRT6 knock-down cells（P<0.05）.9.Similarly,decreased mRNA and protein levels of PPARα were also observed in OSS₁28167 treated cells（P<0.05）.10.The activity of core promoter was inhibited by PPARαknock-down（P<0.05）.11.PPARα silencing resulted in decrease of HBV core DNA and HBV3.5-kb RNA levels,HBsAg and HBeAg secretions,as well as HBs protein expression（P<0.05）.12.PPARα-silencing abolished the increase of HBV core DNA and HBV 3.5-kb RNA levels triggered by SIRT6（P<0.05）.13.Both SIRT6 and PPARα contributed to OSS₁28167 mediated downregulation of HBV core DNA and HBV 3.5-kb RNA levels（P<0.05）.Conclusion:In this paper,we analyzed the effect of SIRT6 inhibitor OSS₁28167on HBV replication.These results showed that OSS₁28167 could inhibit the replication of HBV in HBV stable expression cell lines and HBV natural infection models;In addition,OSS₁28167 also played an effective antiviral role in HBV transgenic mice;Furthermore,we revealed that through inhibiting SIRT6 deacetylase activity,OSS₁28167 downregulated transcription factor PPARα expression,which could enhance HBV core promoter activity.In summary,our results showed that OSS₁28167 might serve as a potential antiviral agent for HBV therapy.