转染杜氏盐藻14-3-3基因对食管鳞癌EC9706细胞Bcl-2和Caspase-9蛋白表达的影响

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

目的:探讨转染杜氏盐藻14-3-3基因对食管鳞癌EC9709细胞Bcl-2和Caspase-9蛋白表达的影响。方法:将杜氏盐藻14-3-3基因cDNA序列分别亚克隆入表达载体pEGFP-C1和pcDNA3.1(+)的HindⅢ和BamHⅠ位点,构建真核表达载体pEGFP-C1-14-3-3和pcDNA3.1(+)-14-3-3,采用脂质体方法转染EC9706细胞,同时设立空载体对照和空白对照,荧光显微镜观察pEGFP-14-3-3融合蛋白在EC9709细胞中的定位;G-418筛选稳定转染pcD-NA3.1(+)-14-3-3的细胞株,采用RT-PCR和Western blotting法检测14-3-3基因的表达;Western blotting检测3种细胞中Bcl-2和Caspase-9蛋白的表达。结果:pEGFP-14-3-3融合蛋白定位于EC9706细胞的细胞核;通过G-418筛选,最终获得稳定表达盐藻14-3-3基因的转染细胞株;与转染空载体和空白对照的EC9706细胞相比,转染pcD-NA3.1(+)-14-3-3的EC9706细胞中Bcl-2蛋白表达量明显增高[(1.60±0.20)、(0.90±0.15)和(2.40±0.10);F=69.8897,P<0.001],而Caspase-9蛋白表达量明显降低[(1.00±0.04)、(1.40±0.08)和(0.40±0.05);F=217.0667,P<0.001]。结论:杜氏盐藻14-3-3基因可能在EC9706细胞凋亡过程中发挥了抑制作用。

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.