RNA干扰Caspase-3基因对镉诱导转化人胚肾293细胞凋亡的影响

Influence of RNA Interference Caspase-3 Gene on HEK 293 Cell Apoptosis Induced by Cadmium

目的研究RNA干扰Caspase-3基因对镉致转化人胚肾293细胞凋亡的影响,探讨Caspase-3基因在镉致细胞凋亡中的作用。方法选择转化人胚肾293细胞进行体外培养,以小干扰RNA(small interference RNA,siRNA)处理细胞36h后,再以40μmol/L氯化镉分别处理12～24h,以四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞活力,以逆转录聚合酶链反应(RT-PCR)和Western-blot法分别检测Caspase-3基因表达水平和蛋白表达水平,以流式细胞仪Annexin-Ⅴ-PI双染法检测细胞凋亡率。结果40μmol/L氯化镉处理转化人胚肾293细胞12h后,Caspase-3基因mRNA水平显著增高,相对分子质量为20kDa的活性亚单位条带显著增强;24h后,细胞凋亡率显著增加(n=4,P<0.01),RNA干扰显著抑制镉诱导的细胞Caspase-3基因mRNA和相对分子质量为20kDa的蛋白表达水平,使细胞凋亡率显著降低(n=4,P<0.01)。结论体外实验表明,RNA干扰Caspase-3基因对镉诱导转化人胚肾293细胞凋亡具有明显的抑制作用,Caspase-3基因在镉诱导转化人胚肾293细胞凋亡过程中起着重要的作用。

Objective To study the influence of RNA interference Caspase-3 gene on apoptosis of transformed human embryonic kidney 293 cells induced by cadmium in vitro. Methods Transformed human embryonic kidney 293 cell were treated with siRNA for 36 h. The treated cells were incubated with 40 μmol/L CdCl2 for 12-24 h and the cells viability were measured with methyl thiazolyl tetrazolium （MTT） assay. In addition, the expression ofcaspase-3 gene in 293 cells was detected by the methods of reverse transcription polymerase chain reaction （RT-PCR） and Western-blot analysis, and the occurrence of apoptosis was determined by flowcytometry with Annexin-V and propidium iodide （PI） double labeling method. Results The results of RT-PCR and Western-blot analysis revealed that in incubated cells with 40 μmol/L cadmium 12 h, the expression of Caspase-3 mRNA and 20 kDa protein significantly increased when compared with controls （n=4, P〈0.01 ）. The apoptosis rate enhanced significantly in incubated cells with 40 μmo]/L cadmium for 24 h （n=4, P〈0.01 ）. RNA interference inhibited the expression of Caspase-3 gene mRNA and 20 kDa protein and decreased the apoptosis rate induced by cadmium. Conclusion RNA interference Caspase-3 gene could significantly inhibit the apoptosis rate induced by cadmium. It was suggested that Caspase-3 gene could play a central role in cadmium-induced cell apoptosis.