title: The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus.
abstract: BACKGROUND
  Hepatitis B virus (HBV) infection correlated with the development of cirrhosis, liver failure and hepatocellular carcinoma (HCC), poses a huge health burden on the global community. However, the pathogenesis of chronic hepatitis B (CHB) remains unclear. Apolipoprotein A1 (ApoA1) mainly secreted by hepatocytes, represents the major protein component of high-density lipoprotein. ApoA1 secretion may be disrupted by HBV infection. In this study, we mainly investigated the molecular mechanism of ApoA1 down regulated by HBV for revealing the pathogenesis of CHB.
  
  
  METHODS
  ApoA1 expression in livers of CHB patients as well as healthy controls were performed by Real-time PCR (RT-PCR) and Western blot. The serum ApoA1 levels were measured by Enzymed-linked immunosorbent assay (ELISA). Expression of ApoA1 mRNA and protein levels were performed by RT-PCR and Western blot in human hepatoma HepG2 cells and subline HepG2.2.15 cells. HBV expression construct, pHBV1.3 were transfected into HepG2, the changes of ApoA1 mRNA and protein expression were detected by RT-PCR and Western blot. To further study the mechanism of ApoA1 down regulation by HBV, 11 CpG islands in ApoA1 promotor were tested for DNA methylation status by MSP. HepG2.2.15 cell lines were treated with DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), then, expression of ApoA1 mRNA and HBV particles in the supernatant, as well as ApoA1 protein levels were detected by RT-PCR and Western blot. Secretion of HBsAg and HBeAg in HepG2 cells cotransfected with pApoA1 and pHBV1.3 constructs was tested by ELISA. Meanwhile, secretion of HBsAg and HBeAg in the supernatant were quantified by ELISA in the HepG2.2.15 cells treated with 5-aza-dC plus ApoA1 siRNA.
  
  
  RESULTS
  Expression of ApoA1 mRNA and protein levels, as well as serum ApoA1 levels in CHB patients were decreased corresponding healthy controls in vivo. In addition, the expression of ApoA1 mRNA and protein levels were down regulated in HepG2.2.15 cells correponding HepG2 cells, 11 CpG islands in ApoA1 promoter were tested for methylation status by MSP in HepG2.2.15 cells compared to HepG2 cells, while two CpG islands were found hypermethylated. Expression of ApoA1 mRNA and protein levels were increased in HepG2.2.15 cells treated with DNA methyltransferase inhibitor 5-aza-dC. Furthermore, overexpression of ApoA1 can enhance HBV expression in HepG2 cells while the inhibitory effect of 5-aza-dC on HBV expression was completely abolished by blocking 5-aza-dC-induced up-regulation of ApoA1 using RNAi.
  
  
  CONCLUSIONS
  Epigenetic silencing of ApoA1 gene expression by CpG island DNA hypermethylation induced by HBV may contribute to the pathogenesis of CHB.
authors.affiliation: School of Biomedical Sciences, Chengdu Medical College, Sichuan, 610500, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: School of Biomedical Sciences, Chengdu Medical College, Sichuan, 610500, China.
authors.forename: Junli
authors.initials: J
authors.lastname: Hao
authors.affiliation: School of Biomedical Sciences, Chengdu Medical College, Sichuan, 610500, China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Liu
authors.affiliation: School of Biomedical Sciences, Chengdu Medical College, Sichuan, 610500, China.
authors.forename: Hongxin
authors.initials: H
authors.lastname: Wang
authors.affiliation: School of Biomedical Sciences, Chengdu Medical College, Sichuan, 610500, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zeng
authors.affiliation: School of Biomedical Sciences, Chengdu Medical College, Sichuan, 610500, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: School of Biomedical Sciences, Chengdu Medical College, Sichuan, 610500, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Pharmacology, Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Sichuan, 610041, China. kuangxi56@163.com.
authors.forename: Xi
authors.initials: X
authors.lastname: Kuang
authors.affiliation: School of Biomedical Sciences, Chengdu Medical College, Sichuan, 610500, China. ztbill@cmc.edu.cn.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
mesh_terms: D016632:Apolipoprotein A-I / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D016022:Case-Control Studies; D018899:CpG Islands; D019175:DNA Methylation; D000077209:Decitabine; D015536:Down-Regulation; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation / Q000187:drug effects; D056945:Hep G2 Cells / Q000187:drug effects / Q000821:virology; D006515:Hepatitis B virus / Q000187:drug effects / Q000472:pathogenicity*; D019694:Hepatitis B, Chronic / Q000235:genetics* / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D011401:Promoter Regions, Genetic
chemical_list: C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D000077209:Decitabine; D001374:Azacitidine
keywords: 5-aza-dC; Apolipoprotein A1; Hepatitis B virus; Hypermethylation