title: Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx.
abstract: BACKGROUND
  Hepatitis B virus (HBV) is one of the major pathogens of human liver disease. Studies have shown that HBV X protein (HBx) plays an important role in promoting viral gene expression and replication. In this study we performed a global proteomic profiling to identify the downstream functional proteins of HBx, thereby detecting the mechanisms of action of HBx on virion replication.
  
  
  METHODS
  HBx in the HepG2.2.15 cell line was knocked down by the transfection of small interfering RNA (siRNA). The replication level of HBV was evaluated by microparticle enzyme immunoassay analysis of HBsAg and HBeAg in the culture supernatant, and real-time quantitative PCR analysis of HBV DNA. Two-dimensional electrophoresis combined with MALDI-TOF/TOF was performed to analyze the changes in protein expression profile after treatment with HBx siRNA.
  
  
  RESULTS
  Knockdown of HBx disturbed HBV replication in vitro. HBx target siRNA significantly inhibited the expression of HBsAg, HBeAg and the replication of HBV DNA. Twelve significantly changed proteins (7 upregulated and 5 downregulated) were successfully identified by MALDI-TOF/TOF using proteomics differential expression analysis after the knockdown of HBx. Among these identified proteins, HSP70 was validated by Western blotting.
  
  
  CONCLUSIONS
  The results of the study indicated the positive effect of HBx on HBV replication, and a group of downstream target proteins of HBx may be responsible for this effect.
authors.affiliation: Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, and Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
authors.forename: Hai-Yang
authors.initials: HY
authors.lastname: Xie
authors.forename: Jun
authors.initials: J
authors.lastname: Cheng
authors.forename: Chun-Yang
authors.initials: CY
authors.lastname: Xing
authors.forename: Jin-Jin
authors.initials: JJ
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Su
authors.forename: Xu-Yong
authors.initials: XY
authors.lastname: Wei
authors.forename: Lin
authors.initials: L
authors.lastname: Zhou
authors.forename: Shu-Sen
authors.initials: SS
authors.lastname: Zheng
mesh_terms: D000704:Analysis of Variance; D015153:Blotting, Western; D003470:Culture Media / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D015967:Gene Expression Regulation, Viral; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics / Q000254:growth & development*; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology*; D006801:Humans; D007124:Immunoenzyme Techniques; D016133:Polymerase Chain Reaction; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015534:Trans-Activators / Q000172:deficiency* / Q000235:genetics; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins; D014779:Virus Replication
chemical_list: D003470:Culture Media; D004279:DNA, Viral; D018840:HSP70 Heat-Shock Proteins; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein