Down-regulated BARF1 expression induces EBV-positive gastric carcino-ma cell apoptosis via activating caspase-dependent mitochondrial pathway / 中国病理生理杂志

ABSTRACT

[ ABSTRACT] AIM:To investigate the effects of BARF1 down-regulation on EBV-positive gastric carcinoma cell apoptosis, and the molecular mechanisms by BARF1 silencing-mediated apoptosis.METHODS: After NUGC3 and SNU719 cells were transfected with NCsiRNA and siRNA, respectively, the protein levels of BARF1, Bcl-2, Bax, cyto-chrome C, caspase 3 and capase 9 were detected by Western blot, and the mRNA expression of BARF1, Bcl-2 and Bax was determined by RT-PCR.The cell viability was measured by the method of Trypan blue exclusion and the cell apoptosis was analyzed by flow cytometry analysis with Annexin V-FITC/PI staining.The expression of the apoptosis-related proteins in the cells transfected with siRNA and NCsiRNA was examined by human apoptosis antibody arrays.Mitochondrial mem-brane potential was determined by flow cytometry.The interaction between Apaf-1 and caspase 9 was confirmed by immuno-precipitation.RESULTS: Compared with untreated and NCsiRNA groups, BARF1 gene silencing significantly inhibited the cell viability, induced apoptosis, and reduced the mitochondrial membrane potential in the NUGC3 and SNU719 cells transfected with siRNA.BARF1 gene silencing up-regulated the expression of pro-apoptotic proteins and down-regulated the expression of anti-apoptotic proteins, and the Bcl-2/Bax ratio was significantly decreased.In BARF1 gene silencing cells, the caspase inhibitor z-VAD-fmk inhibited BARF1 silencing-mediated apoptosis, and significantly increased the levels of cleaved caspase 3 and caspase 9.The concentration of cytochrome C significantly increased as compared with NCsiRNA group, and Apaf-1 interacted with caspase 9 in the cytoplasm.CONCLUSION:BARF1 silencing induces apoptosis via the mitochondrial pathway through regulating the expression of Bcl-2 and Bax proteins in a caspase-dependent manner in the NUGC3 and SNU719 cells.