title: Effect of Hepatitis B Virus X Gene on the Expression Level of p53 Gene using Hep G2 Cell Line.
abstract: BACKGROUND
  The HBV-X (HBX) protein is believed to contribute to the development of HCC. However, the molecular mechanisms involved in HBX- mediated hepatocarcinogenesis remain obscure. In this study, the effect of hepatitis B virus X gene and its protein product HBxAg on expression of p53 gene in Hep G2 cell line was investigated.
  
  
  METHODS
  Viral DNA extracted from HBV-positive serum and HBX gene region was amplified using polymerase chain reaction (PCR). Then, PCR product was cloned into the pcDNA3 vector. After confirmation of cloning, the recombinant plasmid pcDNA3-X was transfected into HepG2 cell line using lipid-mediated DNA-transfection procedure. SDS-PAGE and western blotting methods were used to identify expression of HBX protein. Relative quantification was used to analyze the p53gene expression using the 2-(ΔΔ Ct) method.
  
  
  RESULTS
  Recombinant plasmid pcDNA3-HBX was confirmed by restriction endonucleases digestion and colony-PCR. The results of SDS-PAGE and western blot assays showed that HBX gene could be expressed in Hep G2 cell line. There was no significant difference between the expression levels of p53 compared with GAPDH gene as housekeeping gene (p < 0.05).
  
  
  CONCLUSIONS
  There was no significant difference in the protein levels between the transfected cells with X gene containing HBX130 and HBX131 double mu-tations and p53 gene. It is necessary to do more studies on Hepatitis B virus to understand the role of HBX on the development of liver cancer and its function on p53 tumor suppressor protein.
authors: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran ; Department of Microbiology, School of Biological Science, Shahid Beheshti University, Tehran, Iran.
authors: Roghyeh
authors: R
authors: Kordestani
authors: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran ; Department of Microbiology, School of Biological Science, Shahid Beheshti University, Tehran, Iran.
authors: Hamideh
authors: H
authors: Mirshafiee
authors: Department of Microbiology, School of Biological Science, Shahid Beheshti University, Tehran, Iran.
authors: Seyed Masoud
authors: SM
authors: Hosseini
authors: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
authors: Zohreh
authors: Z
authors: Sharifi
keywords: Hep G2 cell line; Hepatitis B virus; X gene; p53 gene