摘要
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 mediated p38 MAPK gene silencing on mitochondrial pathway of the apoptotic Lovo cells induced by photoexcited TαPc Zn. Methods Chemically synthesized siRNA targeting p38 MAPK gene was transferred into Lovo cells exposed red-light irradiation. The mRNA and protein expressions of p38 MAPK were evaluated with RT-PCR and western blot respectively to decide its interfering efficiency. The effect of photoexcited TαPc Zn on the expression of AIF,Cyto-c,Bcl-2 and Caspase-3,as well as ΔΨmin Lovo cells without or with siRNA-p38 MAPK were evaluated by western blot and JC-I staining respectively. Results The expressions of p38 MAPK mRNA and protein were significantly down regulated in Lovo cells transfected with p38MAPK-targeting siRNA as compared with the control group( P 0. 01). Photoexcited TαPc Zn resulted in apoptosis induction,activation of caspase-3,downregulation of Bcl-2,release of AIF and Cyto-c from mitochondria,and disruption of mitochondria membrane potential in photoexcited TαPc Zn-treated Lovo cells. In contrast,p38MAPK-siRNA attenuated induction of apoptosis,decreased expression of caspase-3,and upregulated expression of Bcl-2,reduced release of AIF and Cyto-c from mitochondria,disrupted the mitochondria membrane potential in photoexcited TαPc Zn-treated Lovo cells. Conclusion p38 MAPK gene silencing could significantly interfere with mitochondrial pathway to weaken the apoptosis of Lovo cells induced by photoexcited TαPc Zn.
出处
《航天医学与医学工程》
CAS
CSCD
北大核心
2016年第4期245-249,共5页
Space Medicine & Medical Engineering
基金
黑龙江省自然科学基金重点项目(ZD201318)