title: [Hepatitis B virus X promotes HepG2 cell cycle progression and growth via downregulation expression of p16 protein].
abstract: OBJECTIVE
  To investigate the effects and related mechanisms of hepatitis B virus X (HBx) protein on cell cycle and growth in hepatocellular carcinoma.
  
  
  METHODS
  A human hepatocyte HepG2 cell line stably expressing a green fluorescent protein (GFP)-tagged HBx (HepG2/GFP-HBx cells) was used for the experiment, and HepG2 parental and HepG2/GFP cells was used as the controls. Effect of HBx on cell growth was evaluated by the MTT cell proliferation assay and on cell cycle progression by flow cytometry analysis of cells with or without treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 5 pmol/L). Effect of HBx expression on promoter methylation status of the p16INK4A tumor-suppressor gene was detected by methylation-specific polymerase chain reaction and on p16 protein level was analyzed with western blotting.
  
  
  RESULTS
  The HepG2/GFP-HBx cells showed significantly higher cell proliferation at 72 hrs of culture (3.225+/-0.038 A490) than either control (HepG2: 2.012+/-0.022 A490, t = -46.86, P less than 0.001; HepG2/GFP: 2.038+/-0.029 A490, t = 42.51, P less than 0.001). The HepG2/GFP-HBx cells also showed significantly lower proportion of cells in the G0/G1 phase (16.45%+/-0.45%) than either control (HepG2: 44.81%+/-1.36%, t = -34.202, P less than 0.001; HepG2/GFP: 42.76%+/-1.58%, t = -28.88, P less than 0.001). However, 5-Aza-CdR treatment did lead to a significant amount of HepG2/GFP-HBx cells being arrested in the G0/G1 phase (33.25%+/-0.79%, t = 31.85, P less than 0.001). The p16INK4A promoter was methylated in the HepG2/GFP-HBx cells, and became demethylation after treatment with 5-Aza-CdR. However, no methylation of p16INK4A promoter was observed in both HepG2 and HepG2/GFP cells. The p16 protein level was significantly lower in the HepG2/GFP-HBx (vs. HepG2 and HepG2/GFP cells) and this level increased after treatment with 5-Aza-CdR.
  
  
  CONCLUSIONS
  HBx protein promotes hepatocellular carcinoma cell cycle progression and growth by shortening the G0/G1 phase, and the underlying mechanism may involve inducing p16INK4A promoter methylation and downregulating p16 protein expression.
authors.affiliation: Department of Infectious Diseases, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China.
authors.forename: Li
authors.initials: L
authors.lastname: Mai
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.forename: Jian-yu
authors.initials: JY
authors.lastname: Kuang
authors.forename: Jian-yun
authors.initials: JY
authors.lastname: Zhu
authors.forename: Yan-hong
authors.initials: YH
authors.lastname: Kang
authors.forename: Fu-cheng
authors.initials: FC
authors.lastname: Zhang
authors.forename: Qi-feng
authors.initials: QF
authors.lastname: Xie
authors.forename: Zhi-liang
authors.initials: ZL
authors.lastname: Gao
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D019942:Genes, p16; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000494:pharmacology*; 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