RNA干扰抑制BAG-1基因表达对皮肤鳞状细胞癌增殖及凋亡的影响研究

摘要

目的:探讨抑制BAG-1(Bcl-2-associated athanogene-1)基因表达对皮肤鳞状细胞癌增殖及凋亡的影响.方法:采用LipofectamineTM2000将BAG-1的siRNA转染皮肤鳞状细胞癌A431,转染后48 h,RT-PCR检测BAG-1的mRNA表达, Western blot检测BAG-1的蛋白表达;CCK8和流式细胞仪分别检测细胞增殖和凋亡情况;Western blot检测B细胞淋巴瘤/白血病-2(Bcl-2)家族促凋亡蛋白 Bcl-2相关X蛋白(Bax)及Wnt/β-catenin信号通路β-连环蛋白(β-catenin)、Survivin的蛋白表达;ELISA试剂盒检测白介素6 (IL-6)、血管内皮生长因子(VEGF)含量.结果:与空白组和阴性对照组比较,转染BAG-1的siRNA可明显抑制BAG-1在转录和翻译水平上的表达;与空白组比较,BAG-1-siRNA组细胞增殖显著降低,细胞凋亡率显著增加,Bax蛋白表达上调,β-catenin、Survivin蛋白表达下调,IL-6、VEGF含量均显著降低(P<0.05).结论:BAG-1表达沉默可降低皮肤鳞状细胞癌增殖,促进细胞凋亡,上调Bax及下调Wnt/β-catenin信号通路表达,降低IL-6、VEGF因子的分泌.%Objective:To investigate the effect of inhibiting of BAG-1 gene expression on proliferation and apoptosis of cutaneous squamous cell carcinoma.Methods:siRNA of BAG-1 was transfected in cutaneous squamous cell carcinoma A431 by Lipo-fectamineTM2000,cells were transfected for 48 h,the BAG-1 mRNA expression was detected by RT-PCR,BAG-1 protein expression was detected by Western blot;CCK8 and flow cytometry were used to detect the cell proliferation and apoptosis;Western blot was used to detect the expression of Bax,β-catenin and Survivin protein;IL-6 and VEGF content were detected by ELISA kit.Results:Compared with control group and negative control group,transfection of BAG-1 with siRNA could significantly inhibit the expression of BAG-1 at the transcriptional and translational levels;compared with the control group,the cells proliferation in BAG-1-siRNA group significantly decreased,apoptosis rate was significantly increased,Bax protein expression was up-regulated,expression of β-catenin,Survivin protein were down regulated,the contents of IL-6 and VEGF were decreased significantly(P<0.05).Conclusion:Silencing of BAG-1 expression can reduce the proliferation of cutaneous squamous cell carcinoma,promote apoptosis,up regulate Bax expression and down regulate the expression of Wnt/β-catenin signaling pathway,and reduce the secretion of IL-6 and VEGF factors.