Effects Of Hepatitis B Virus X Gene On Hepatic Teatosis And Its Mechanism

Background and objectives:Recent years, it is extensive attention about the relationship betweennon-alcoholic fatty liver disease (NAFLD) and viral hepatitis by scholars.Many studies displayed that infection of hepatitis B virus (HBV) andhepatitis C virus (HCV) was always associated with varying degrees ofhepatic steatosis. China is a big country of chronic hepatitis B (CHB), andHBsAg-positive people accounted for7.18%of our population, about120million people, affecting people's living standards seriously. It has definiterelations between chronic hepatitis C (CHC) and hepatic steatosis, butrelations between CHB and hepatic steatosis are still unclear. Some studiesreported host metabolic factors were major reasons of HBV-associated fattyliver, and had little contact with the viral itself. While other researchesbelieved HBV was a major factor leading to fatty liver. It has become a newchallenge to investigate the relationship between HBV infection and hepaticsteatosis and its mechanism in the field of liver diseases research.Hepatitis B virus X protein, a multifunctional protein of the HBx geneencoding, plays an important role in viral replication. Foreign studies had shown that HBx could regulate the transcription of lipid metabolism relatedgenes by interacting with liver X receptors (LXRs) in order to achieve theregulation of hepatic lipid metabolism. LXRs are members of nuclearreceptor superfamily. It is a key nuclear receptor in lipid metabolism, andwidely participates in the body's regulation of lipid metabolism, cholesterolmetabolism and other physiological activities. HBV-related diseases areserious public health issue in our country, and study of HBV and fatty liverhas just started in China. The incidence of CHB-related fatty liver, riskfactors of CHB-related fatty liver, hepatic steatosis on the progression ofCHB and relationship of the response to antiviral therapy, and manyunknown issues must be answered.HepG2.2.15cells, the hepatoma cell line which was integratedHBV-DNA into HepG2cells, can express HBx continuous. It is an importantcell model of HBV study. Whether HBV participates in hepatic steatosis ornot, and what is the effect and mechanism are the focus of this subject. Theresults of this study will help us judge the relationship between HBV andNAFLD, study some knowledge of its mechanism, and provide a theoreticalbasis for HBV related fatty liver therapy.This experiment was to test expressions of HBx and LXRα inHepG2.2.15cells and HepG2cells, construct and filter out the best effectiveshHBx and shLXRα RNA interference plasmids. Then expressions of HBxand LXRα gene would be silenced respectively by using RNA interference technology. Cells were stimulated by using sodium oleate or LXRα agonistT0901317to study the effect of HBx on hepatic steatosis and expressions oflipid metabolism factors, explore the relationship of HBx-LXRα pathway. Atlast, it would be expected to reveal the relationship between HBV infectionand hepatic steatosis from the molecular biology.Methods:1) The expressions of HBx and LXRα mRNA and protein inHepG2.2.15and HepG2cells were detected by using RT-PCR and Westernblot.2) The HBx gene-specific interference plasmid was constructed and thedegree of hepatic steatosis and the expressions of lipid metabolism relatedfactors would be tested after HBx was silenced.3) The LXRα gene-specific interference plasmid was constructed toinvestigate the effect of HBx on liver cells' lipid metabolism and therelationship with LXRα pathway after gene silenced.Results:1) Expressions of HBx and LXRα could be detected in HepG2.2.15cells, but expression of HBx in HepG2cells was not detected. Comparedwith HepG2.2.15cells, expression of LXRα in HepG2cells was lower, andthe difference was statistically significant(P＜0.05).2) The HBx gene specific interference plasmid could be constructedsuccessfully and filtered out the best effective sequences to inhibit HBx expression. Compared with empty control group and negative control group,expressions of HBx mRNA and protein were significant decreased in HBx1,HBx2and HBx3group (P＜0.05), and HBx3group had the lowestexpression after HBx gene silenced.3) Without adding sodium oleate, lipid droplets, expressions ofSREBP-1c mRNA, LXRα and FAS protein in shHBx3transfection groupand HepG2group were significant lower than empty control group andnegative control group after HBx gene was silenced(P＜0.05), and therewas no significant difference between shHBx3transfection group andHepG2group(P＞0.05). While added sodium oleate after HBx gene wassilenced, the content of TG, expression of SREBP-1c mRNA, HBx and FASprotein increased as stimulated time extended, and its expressions in shHBx3and HepG2groups were significantly decreased as compared with emptyand negative control groups at same time point(P＜0.05). There was nosignificant difference between shHBx group and HepG2group at same timepoint.4) The LXRα gene specific interference plasmid could be constructedsuccessfully and filtered out the best effective sequences to inhibit LXRαexpression. Compared with empty control group and negative control group,expressions of LXRα mRNA and protein were significant decreased inLXRα1, LXRα2and LXRα3group(P＜0.05), and LXRα1group had thelowest expression after LXRα gene silenced. 5) Without adding agonist, lipid droplets, expressions of SREBP-1cmRNA, LXRα and FAS protein in shLXRα1transfection group weresignificant lower than empty control group and negative control group afterLXRα gene silenced(P＜0.05), and there was no significant difference ofHBx expression in each group(P＞0.05). While added agonist after LXRαgene silenced, the content of TG, expression of SREBP-1c mRNA, HBx andFAS protein increased as stimulated time extended, and there was nosignificant difference of HBx in each group at the same time point, but theTG content, SREBP-1c, FAS in shLXRα1group were significantlydecreased as compared with empty and negative control groups at same timepoint(P＜0.05).Conclusions:1) Compared with HepG2.2.15cells which expressed HBx gene,expressions of LXRα mRNA and protein in HepG2cells were lower thanthat of in HepG2.2.15cells.2) HBx was one of an important viral factor which caused hepaticsteatosis and participated in regulation of hepatocyte lipid metabolism viaLXRα/SREBP-1c/FAS pathway.3) HBx increased the role of hepatic steatosis which was caused bysodium oleate and T0901317. It suggested that HBV infection is a riskfactor of fatty liver.