皮肤鳞状细胞癌细胞中BAG-1表达变化对细胞增殖、凋亡的影响及机制

Effects of BAG-1 expression on cell proliferation and apoptosis in cutaneous squamous cell carcinoma

目的观察Bcl-2结合抗凋亡基因1(BAG-1)在皮肤鳞状细胞癌(CSCC)细胞株A431中的表达水平,并探讨其对A431细胞增殖和凋亡的影响及其作用机制。方法分别采用qRT-PCR及Western blotting法检测CSCC细胞株A431和人正常永生化上皮细胞Hecate中BAG-1 mRNA、蛋白表达。常规培养CSCC细胞株A431,感染BAG-1敲减病毒(抑制组,sh-BAG-1),并设立阴性对照(对照组),培养48 h后,分别采用qRT-PCR及Western blotting法检测BAG-1 mRNA及蛋白的表达,采用MTS法检测细胞增殖活力、平板克隆试验检测细胞克隆形成能力、流式细胞术检测细胞周期和细胞凋亡率、Western blotting法检测Mdm-2、p53及其下游基因Bcl-2和p21蛋白的表达水平。结果 CSCC细胞系A431中BAG-1 mRNA相对表达量高于人正常永生化上皮细胞Hecate(P<0.05)。与对照组比较,抑制组BAG-1蛋白相对表达量下降,细胞增殖及克隆形成能力减弱,细胞阻滞在G1期,细胞凋亡增多,Mdm-2蛋白表达减少,p53、p21和Bcl-2蛋白表达均增多(P均<0.05)。结论 CSCC细胞中BAG-1高表达,其可能通过调控Mdm-2/p53信号通路增强CSCC细胞增殖、抑制其凋亡。

Objective To observe the expression of Bcl-2combined with anti-apoptosis gene1(BAG-1)in the cutaneous squamous cell carcinoma(CSCC)A431cells,and to explore its effects on A431cell proliferation and apoptosis and its mechanism.Methods The expression of BAG-1mRNA and protein in CSCC A431cells and Hecate of human normal immortalized epithelial cells was detected by qRT-PCR and Western blotting,respectively.CSCC A431cells were cultured routinely and infected with BAG-1knockdown virus(inhibition group,sh-BAG-1),and the negative control group was also established.After48-hour culture,the expression of BAG-1mRNA and protein was detected by qRT-PCR and Western blotting,respectively.MTS method was used to detect the cellular proliferative activity,plate clone test was used to detect cell cloning formation ability,and flow cytometry was used to detect cell cycle and apoptotic rate.The expression of Mdm-2,p53and their downstream genes Bcl-2and p21protein was detected by Western blotting.Results The relative expression level of BAG-1mRNA in the CSCC A431cells was higher than that in Hecate of human normal immortalized epithelial cells(P<0.05).Compared with the control group,the relative expression of BAG-1protein in the inhibition group decreased,the cell proliferation and cloning ability decreased,and the cells arrested in the G1phase,the apoptosis increased,the expression of Mdm-2protein decreased,and the expression of p53,p21,and Bcl-2protein increased in the inhibition group(all P<0.05).Conclusion The BAG-1in the CSCC cells is highly expressed,which may enhance the proliferation and inhibit apoptosis of CSCC by regulating Mdm-2/p53signaling pathway.