| Objective:To study the effect of BARF1 gene encoded by Epstein-Barr virus (EBV) in gastric cancer cell on cell biological behavior so as to know the function of BARF1 protein and the role in EBV-associated gastric carcinoma(EBVaGC).Methods:1) BARF1 open reading frame was amplified by polymerase chain reaction(PCR) based on the DNA templete extracted from EBV positive cell line B95-8 and cloned into pSG5 eukaryotic expression vector through pGEM-T cloning vector and confirmed by restriction enzymes and DNA sequencing.2) pSG5-BARF1 and pSG5 vector control were separately transfected into moderately differentiated gastric cancer cell line SGC7901.The transfected cells were cloned by limited dilution and selected by geneticin G418.BARF1 exprssion was confirmed by RT-PCR.3) The effect of BARF1 on cell proliferation was carried out by cell counting and MTT assay.The function on apoptosis induced by anti-cancer drug diamminedichloroplatinum(DDP) was preformed by cell counting,Hoechst 33342 staining and DNA ladder assay in agar gel electrophoresis.The carcinogenicity was determined by soft-agar colony formation assay and the cell migration ability by transwell migration assay.Results:1) pSG5-BARF1 was successfully constructed and transfected into SGC7901 cells and cell clones stably expressing BARF1 were obtained.2) There were higher proliferation velocities,higher survival rate and lower apoptosis rate and less DNA ladder after induction by DDP,higher colony formation rate in soft agar and higher migration rate in BARF1 expressing clones comparing to the control.Conclusion:Our result indicated that BARF1 expression in gastric carcinoma cells led to more resistant to apoptosis induced by DDP,faster proliferation,higher colony formation rate,and stronger migration capability,and suggest that BARF1 gene has all around oncogenic ability in gastric epithelial cells and may play an important role in EBV-associated gastric carcinoma. |
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