Sirt1-Mediated Synthesis Of Triglyceride By HCV Core Protein In HepG2 Cells

[Objectives]To investigate on regulation mechanisms of Sirt1 mediated HCV core protein on triglyceride accumulation.[Methods]1.By Real-time PCR,Western blot and adipogenesis assay kit,analyze that HCV core protein could promote the liver triglyceride accumulation.2.By small interfering RNA and short hairpin RNA(shRNA),to explore the HCV core protein on lipid metabolism related gene regulation after interference Sirtl.3.By Cre/Lox P system,knockout mice of Sirtl are bred successfully.4.To construct HCV core protein(1b/3a)lentiviral vector pCDH-CMV-MCS-EF1-copGFP-HCV core and pack lentivirus of HCV core[Results]1.The results of quantitative determination of triglyceride showed that overexpression of pcDNA3.1/myc-His-core1b/3 a,HCV core protein can significantly increase the triglyceride content of HepG2 cells.2.The results of real-time PCR showed that HCV core protein can increase lipid synthesis related genes(SREBP1c,FASN,PPARy)mRNA level in HepG2 cells.3.According to the results of Western blot,HCV core protein can increase lipid synthesis related genes(SREBP1,PPAR?)protein levels in HepG2 cells.4.The results of Western blot of interference Sirtl showed that the protein levels of the genes(SREBP1,FASN)related to lipid synthesis were down-regulated after interfering Sirtl successfully.5.Knockout mice of Sirtl are bred successfully.6.The lentiviral vector pCDH-CMV-MCS-EF 1-copGFP-HCV core(1b/3a)were constructed and lentivirus of HCV core were packed successfully.7.Construct HCV core protein(lb/3a)lentiviral vector pCDH-CMV-MCS-EF1-copGFP-HCV core(1b/3a)and pack lentivirus of HCV core successfully.[Conclusions]In vitro,pcDNA3.1/myc-His-core was expressed successfully in HepG2 cells,compared with the group of transfection empty vector pcDNA3.1/myc-His(-),we can conclude that HCV core can promote lipid synthesis and its related gene transcription level and protein expression level in core-expreession HepG2 cells.After interference Sirtl,overexpression of HCV core at the same time,the protein expression level of lipid related gene was down-regulated.So we can come to a conclusion that HCV core regulates lipid synthesis via Sirt1-mediated in HepG2 cells and promote the synthesis of lipids.In addition,knockout mice of Sirt1 are bred successfully.The lentiviral vector pCDH-CMV-MCS-EF1-copGFP-HCV core(1b/3a)were constructed and lentivirus of HCV core were packed successfully.