Bile Acid Receptor FXR Regulates The Expression And Secretion Of HBV

BackgroundHepatitis B virus was a partially double-stranded DNA virus,about 350 million people were infected in worldwide.Chronic persistent infection could cause chronic hepatitis,liver cirrhosis and liver cancer.HBV infection has tissue specificity and species specificity.The hepatotropism of HBV infection was associated with liver specific receptors and live transcription factors.Previous study has found a functional receptor of the HBV infection,which was Na~+/Taurocholate Cotransporting Polypeptide(NTCP).Encoded by SLC10A1 gene,NTCP was a hepatocyte surface receptor protein distributed in the basement membrane of hepatocyte.Moreover,NTCP was not only an important receptor of bile acid enterohepatic circulation in hepatocytes,but also the receptor of HBV turned into hepatocytes.The inhibition of NTCP function induced by HBV infection could lead to a series of changes in the metabolism of cholesterol and bile acid.Farnesoid X receptor(FXR)belongs to a member of the nuclear receptor superfamily,and bile acid was its endogenous ligand.Once activated by bile acid,FXR would regulate a series of genes expression,which played an important role in lipid metabolism,glucose metabolism,bile acid metabolism,liver protection,atherosclerosis,the growth of intestinal flora and innate immune.Patients with acute HBV infection will demonstrate increased bile acid synthesis and total bile acid levels,consisted with the trend of serum transaminases,and the CDCA treatment was basic invalid for HBV,so bile acid in the role of HBV infection had been ignored,and was only used as a diagnostic index.With the discovery of HBV receptor,more researches focused on the relationship between bile acid and HBV infection.However,the mechanisms of bile acid in HBV replication expression and secretion process and chronic persistent infection were less clear.This study would preliminary discuss the role of FXR in the expression and secretion of HBV.ObjectiveThis study would explore the relationship between FXR and transcript expression and secretion of HBV.Methods(1)Building HBV enhancer/promoter luciferase report plasmid pGL3-EN2/Cp with the methods of PCR,double enzyme digestion with SacI and SmaI,purification,connection,transformation and sequencing.(2)In HepG2 cells,cotransfecting pAAV/HBV1.2 and pFXR/pGFP plasmids with the same transfection efficiency and doses.After stimulating 24h and 48h by CDCA,collecting intracellular protein and cell supernatant that were applied to detect HBsAg and HBeAg expression levels with ELISA.(3)In HepG2 cells,cotransfecting pHY106+wta and pFXR/pGFP plasmids with the same transfection efficiency and doses.After stimulating 48h by CDCA,collecting intracellular protein and cell supernatant that were applied to detect HBsAg and HBeAg expression levels with ELISA.(4)In HEK-293T cells,cotransfecting pAAV/HBV1.2 and pFXR/pGFP plasmids with the same transfection efficiency and doses.After stimulating 48h by CDCA,collecting intracellular protein and cell supernatant that were applied to detect HBsAg and HBeAg expression levels with ELISA.(5)In HepG2 cells,cotransfecting pAAV/HBV1.2,and pFXR/pGFP plasmids.After stimulating 48h by CDCA.Total RNA was extracted from the HepG2 cells.Using RT-PCR to detect the expression of preC/pgRNA.(6)In HepG2 cells,cotransfecting PGL3-EN2/Cp and pRL-TK/pFXR plasmids.After stimulating with CDCA for 48h,through the dual luciferase reporter gene detection,analyzing the mechanism of FXR in HBV transcription regulation.(7)Statistical analysis and drawing.Experimental results were analyzed by SPSS17.0 software and t-test was performed.P<0.05 was considered as statistically significant.The drawing was made by GraphPad Prism 5 software.Results(1)pGL3-EN2/Cp plasmid was successfully constructed by sequencing and sequence alignment.(2)In HepG2 cells,after cotransfected with pAAV/HBV1.2 and pFXR/pGFP plasmids,and stimulated with CDCA for 24h or 48h.FXR significantly promoted the expression and secretion of HBeAg(P<0.05);although FXR increased intracellular HBsAg expression(P<0.05),the supernatant HBsAg level didn't rise(P>0.05).(3)In HepG2 cells,after cotransfected with pHY106+wta and pFXR/pGFP plasmids,and stimulated with CDCA for 48h,FXR significantly promoted the expression of HBeAg(P<0.05).(4)In HEK-293T cells,after cotransfected with pAAV/HBV1.2 and pFXR/pGFP plasmids,and stimulated with CDCA for 48h,FXR significantly promoted the expression and secretion of HBsAg and HBeAg(P<0.05).(5)In HepG2 cells,cotransfecting pAAV/HBV1.2,RT-PCR analysis showed that FXR could promote the expression of preC/pgRNA(P<0.05).(6)Dual luciferase report analysis revealed that FXR combining with HBV core promoter EN2/Cp could significantly improve the activity of firefly luciferase(P<0.05).ConclusionsHBV infection results in bile acid rise.After activated bile acid receptor FXR,which will regulate HBV transcript expression and secretion process,this study preliminary obtained the following conclusions:(1)In HepG2 cells,FXR promoted the expression and secretion of HBeAg.(2)In HepG2 cells,FXR promoted the expression of HBsAg,and the secretion of extracellular HBsAg were no difference.FXR relatived to inhibit the secretion of HBsAg.(3)Dual luciferase report analysis and RT-PCR showed that FXR promoted the expression of HBV in transcription level.