title: [Hepatitis B virus X protein induces malignant transformation tendency of Chang liver cells involving p16(INK4a) hypermethylation].
abstract: OBJECTIVE
  To study the relationship between hepatitis B virus X (HBx) protein and DNA methylation of p16(INK4a) and the role of HBx in the  carcinogenesis of hepatocellular carcinoma.
  
  
  METHODS
  Eukaryonic expression vectors pcDNA3.1B-HBx and pcDNA3.1B were transduced into Chang  liver cells by using Lipofectamine 2000 to establish the Chang-HBx liver cell line (HBx expression) and Chang-vector liver cell line (non-HBx  expression). RT-PCR and Western blot were used to test the expression of p16(INK4a) in the two cell lines. The level of p16(INK4a) promoter methylation  was tested by methylation specific PCR (MSP). The proliferation curves were drew by CCK-8, and S-phase in cell cycle and apoptosis were observed by  flow cytometry.
  
  
  RESULTS
  Hypermethylation of p16 can be mediated by HBx, which decreases the expression of mRNA and protein of p16. Chang-HBx  cells grow faster. Chang-HBx cells have much higher S-phase population (28.96% vs. 21.53%, P<0.001; 28.96% vs. 21.5%, P<0.001) and lower apoptosis  rate (2.71% vs. 3.69%, P<0.001; 2.71% vs. 3.36%, P<0.001) than Chang-vector cells and Chang cells respectively.
  
  
  CONCLUSIONS
  p16(INK4a) expression  was repressed by HBx protein via DNA methylation of p16(INK4a), which can induce the malignant transformation tendency of Chang cells.
authors.affiliation: Department of Hepatobilliary Surgery, Hainan Branch of Chinese People's Liberation Army General Hospital, Sanya, China.
authors.forename: Wen-ping
authors.initials: WP
authors.lastname: Lv
authors.forename: Kai-lun
authors.initials: KL
authors.lastname: Zhou
authors.forename: Bin
authors.initials: B
authors.lastname: Gao
authors.forename: Yong-liang
authors.initials: YL
authors.lastname: Chen
authors.forename: Xin
authors.initials: X
authors.lastname: Xiang
authors.forename: Ming
authors.initials: M
authors.lastname: Su
authors.forename: Jia-hong
authors.initials: JH
authors.lastname: Dong
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000821:virology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D019694:Hepatitis B, Chronic / Q000235:genetics / Q000473:pathology / Q000821:virology; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein