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目的探讨RNAi沉默p38MAPK基因表达对光激发TαPc Zn诱导的人结肠癌Lovo细胞线粒体凋亡途径干扰的机制。方法运用siRNA靶向干扰p38MAPK基因,采用RT-PCR和Western blot法分别检测细胞内p38MAPK的干扰效率,同时用Western blot法检测AIF、Bcl-2、Cyto-c及Caspase-3的表达,JC-I染色检测沉默p38MAPK基因对ΔΨm的影响。结果与对照组比较,光作用下siRNA-p38MAPK转染组中p38MAPK的mRNA和蛋白水平显著下调(P<0.01)。在光激发下,TαPc Zn明显诱导Lovo凋亡,ΔΨm降低,从线粒体释放到细胞质的AIF、Cyto-c增加,Bcl-2表达减少,同时caspase-3发生断裂激活。而在siRNA沉默p38MAPK后,光激发TαPc Zn诱导细胞凋亡的作用减弱,ΔΨm有所增加,细胞质中AIF、Cyto-c释放减少,caspase-3激活减弱,Bcl-2表达增加。结论光激发TαPc Zn作用下,沉默p38MAPK基因可明显干扰线粒体途径,进而减弱光激发TαPc Zn所诱导的细胞凋亡。
Objective To investigate the mechanism of RNAi silencing p38MAPK gene expression in light-induced TαPc Zn-induced mitochondrial apoptosis pathway in human colon cancer Lovo cells. Methods The interference of p38MAPK gene with siRNA was detected by RT-PCR and Western blot. The expression of AIF, Bcl-2, Cyto-c and Caspase-3 was detected by Western blot. I staining was used to detect the effect of silenced p38MAPK gene on ΔΨm. Results Compared with the control group, the p38MAPK mRNA and protein levels in siRNA-p38MAPK transfected group were significantly decreased (P <0.01). Under photoexcitation, TαPc Zn significantly induced Lovo apoptosis, decreased ΔΨm, increased release of Cyto-c, decreased Bcl-2 expression, and cleaved activation of caspase-3 from mitochondria to cytoplasmic AIF. After p38MAPK siRNA silencing, the effect of light-induced TαPc Zn-induced apoptosis was weakened, ΔΨm was increased, the release of AIF and Cyto-c in the cytoplasm was decreased, the activation of caspase-3 was decreased and the expression of Bcl-2 was increased. Conclusion Silencing of p38MAPK gene can significantly interfere with the mitochondrial pathway by photoexcitation of TαPc Zn, and then attenuate the apoptosis induced by photoexcited TαPc Zn.