CLIC4在饥饿诱导神经胶质瘤细胞自噬中的作用

The role of CLIC4 protein in autophagy induced by starvation in glioma cells

目的通过饥饿诱导人神经胶质瘤细胞发生自噬,利用siRNA技术部分沉默细胞内氯通道蛋白4(CLIC4),探讨CLIC4在细胞自噬中的作用。方法根据CLIC4的基因序列构建CLIC4siRNA质粒,建立稳定转染CLIC4siRNA的U251细胞株,分析CLIC4蛋白表达;MTT法检测转染pSH1Si-CLIC4对细胞生存率的影响;利用Western Blot检测转染CLIC4siRNA在U251细胞凋亡相关蛋白Bax、Bcl-2表达;检测饥饿条件下U251细胞LC3蛋白、Bax、Bcl-2的表达;检测抑制CLIC4表达对于饥饿条件下细胞白caspase-3和胞浆cytc以及LC3的表达。结果 Western Blot显示,与空质粒对照组相比,转染CLIC4siRNA的U251细胞CLIC4表达显著下降;与对照组相比,转染空质粒细胞组以及瞬时转染pSH1Si-CLIC4载体细胞组U251细胞生存率均无明显差异;单纯CLIC4siRNA对于U251细胞Bax、Bcl-2表达无影响;与对照组相比,饥饿8h能够诱导U251细胞自噬,LC3水平显著增加,而细胞Bax、Bcl-2表达无明显变化;转染CLIC4siRNA后饥饿8h,caspase-3、cytc以及LC3表达增加。结论单纯CLIC4siRNA对于U251细胞自噬和凋亡均无显著影响,饥饿条件下U251细胞发生自噬的同时并没有明显细胞凋亡过程,抑制CLIC4表达促进了饥饿条件下的U251细胞自噬,同时能够启动线粒体相关途径的细胞凋亡。

Objective To investigate the mechanism and effects of CLIC4 protein in glioma cells autophagy induced by starvation through siRNA silencing CLIC4. Methods Based on the gene sequence,CLIC4 siRNA plasmid and stable transfected U251 cells were constructed. The level of CLIC4 expression was analyzed. To detect cell vitality, MTT method was applied. To investigate the effect of CLIC4 siRNA on the apoptosis induced by starvation, the levels of Bax, Bcl-2 were detected. To investigate the effect of CLIC4 siRNA on the apoptosis and autophagy induced by starvation, the levels of cleaved caspase-3,cytosolic cyt c and LC3 were detected. Results Compared with the control group,the results indicated that there was obvious decrease of CLIC4 expression in U251 cells with CLIC4 siRNA transfected. MTT results indicated that there was no obvious effect of cell vitality with CLIC4 siRNA transfected There were no obvious changes in protein Bax and Bcl-2 with CLIC4 siRNA transfected U251 cells. Compared with control group, the level of LC3 had a significant increase under starvation in U251 cells at 8 hours. Meanwhile,protein Bax,Bcl-2 had no significant change under starvation. Western Blot indicated cleaved caspase-3, cytosolic cyt c and LC3 expression increased in CLIC4 siRNA transfected U251 cells under starvation. Conclusion The results showed that CLIC4 siRNA had no effect on cell autophagy and apoptosis. Under starvation, autophagy was detected in U251 cells but no apoptosis. CLIC4 siRNA enhanced the autophagy under starvation,on the same time, triggered caspase-dependent mitochondrial apoptosis.