Expression of Bcl-2 and Caspase-9 proteins of EC9706 cell transfected 14-3-3 gene of Dunaliella salina

Aim:To investigate the effect of the 14-3-3 gene of Dunaliella salina on the expression of Bcl-2 and Caspase-9 proteins of esophageal squamous cell cancer cell line EC9706.Methods:The cDNA fragment of the 14-3-3 gene of Dunaliella salina was subcloned into the HindⅢ-BamHⅠsites of the eukaryotic expression vector pEGFP-C1 and pcDNA3.1(+),respectively,generating recombinant expression vectors pEGFP-C1-14-3-3 and pcDNA3.1(+)-14-3-3.The recombinant vectors were transfected into EC9706 cells by LipofectamineTM 2000 at the same time,the vectors without 14-3-3 genes served as controls,the location of the fusion EGFP-14-3-3 proteins was determined by fluorescent microscope and the EC9706 cells transfected with pcDNA3.1(+)-14-3-3 were selected with G-418 and characterized by RT-PCR and Western blotting,then the expression of the Bcl-2 and Caspase-9 proteins in the transfectants was monitored by Western blotting.Results:pEGFP-14-3-3 fusion proteins were localized in the nucleus of EC9706 cells.EC9706 cells stably expressing the 14-3-3 gene of Dunaliella salina were obtained with G-418 selection.The expression of Bcl-2 protein significantly increased[(1.60±0.20),(0.90±0.15) and(2.40±0.10);F=69.889 7,P0.001] while that of Caspase-9 protein decreased[(1.00±0.04),(1.40±0.08) and(0.40±0.05);F=217.066 7,P0.001] in the EC9706 cells transfected with pcDNA3.1(+)-14-3-3,compared with the control groups.Conclusion:The introduction of the 14-3-3 gene of Dunaliella salina upregulates the expression of Bcl-2 protein while downregulates that of Caspase-9 protein in the EC9706 cells,suggesting its potential role in inhibiting the apoptosis of cells.