Regulation Of Hepatitis B Virus S Protein On Human APOA? Gene

Hepatitis B virus(HBV)infection can cause acute and chronic hepatitis B,liver cirrhosis,and is closely related to the occurrence and development of primary hepatocellular carcinoma(HCC).However,its pathogenic mechanism is not yet clear.HBs protein is an HBV-encoded envelope protein involved in the assembly of intact HBV Dane particles,the assembly of spherical and secreted filaments,and is abundantly present in the serum of HBV-infected individuals.Studies have found that HBs could affect cell physiological functions,such as carbohydrate and lipid metabolism,cell growth and apoptosis.Our presious study found that apoA was down-regulated in HBV stable expression cell line HepG2.2.15 compared with control cell line HepG2 using real-time PCR,suggesting that HBV can reduce apolipoprotein AII expression in mRNA level.To further understand the regulated mechanisms of HBs in APOA II can help understand the pathogenicity of HBV.The first part of this study aims to verify the effect of HBV,especially HBs,on the transcriptional level APOAII.First,the 1.2 × HBV genomic DNA plasmid was transfected in HepG2 cell lines and real-time PCR and Western blot were used to detect the expression of APOAII.Then HBs,the protein of HBV was transfected in HepG2 cells through adenovirus system.After that,real-time PCR and Western blot were used to detect the expression of APOAII,confirming whether HBV and HBs can down-regulate APOAII gene transcription and expression.The second part of this study was to elucidate the mechanisms underlying the down-regulation of APOAII gene transcription and expression by HBs.In this section,we first constructed the full-length promoter region of APOAII(-2700 nt ~ +50 nt)and segmented reporter gene recombinant expression vector.The results showed that the-1029 nt ~ +50 nt region retains the maximum activity of APOAII promoter.A site-by-site deletion of the cis-acting elements that may exist in the maximal active region of the apoAII promoter(a total of 14 elements in the A-N)was cloned into the pGL4.10 vector to construct a cis-acting element deleted recombinant vector and transfected into HepG2 cells.The activity of luciferase was found to be A-component(-40 nt ~-33nt),B-element(-65 nt ~-42nt),C-element(-126 nt ~-110nt),H-element(-573 nt ~-554nt),the K element(-760 nt to-743 nt),and the L element(-803 nt to-773 nt)are the key active elements on the APOAII promoter.On this basis,cells were infected with Ad-HBs or Ad-GFP and luciferase screened for cis-acting elements specifically regulated by HBs.The results showed that K-elements were the key components for the down-regulation of APOAII expression by HBs.Using TFSEARCH and TESS online software to predict potential transcription factor binding sites in the promoter region of K element,it was found that K element(-760 nt ~-743nt)region contains hepatocyte nuclear factor 4?(HNF4?),CCAAT/enhanced Binding protein beta(CCAAT/enhancer Binding protein ?,C/EBP?),cAMP responsive element binding protein(CREB).Further construction of C/EBP?,C/EBP?,and CREB overexpression vectors transfected into HepG2 cells can down-regulate APOAII transcription.Construction of HNF4? over-expression vector can up-regulate APOAII transcription.On this basis,gel retardation experiments and chromatin immunoprecipitation experiments showed that HNF4?,C/EBP?,C/EBP?,and CREB specifically bind to the promoter region of APOAII K element.To further verify the effect of HBs on potential transcription factors,real-time RT-PCR and western blot were used to screen which transcription factors were specifically regulated by HBs infection with Ad-HBs or Ad-GFP.The results showed that HBs could up-regulate the transcription and expression of C/EBP?,C/EBP?,CREB and down-regulate the transcription and expression of HNF4?.Therefore,we conclude that HBs downregulates the transcription and expression of APOAII by up-regulating C/EBP?,C/EBP?,CREB,down-regulating HNF4? transcription and expression.