Cofilin-1 siRNA的设计及其对细胞自噬的影响

Knock-down of Cofilin-1 by siRNA and its effect on cellular autophagy

目的设计Cofilin-1的小分子干扰RNA(siRNA),并利用该方法初步探索干扰Cofilin-1对细胞自噬的影响。方法利用在线软件及根据文献报道设计出3对靶向人Cofilin-1的siRNA,转染人胚肾细胞HEK293,蛋白免疫印迹法检测细胞Cofilin-1、p62、LC3Ⅰ及LC3Ⅱ蛋白的表达变化。mRFP-GFP-LC3双荧光观察细胞自噬流(自噬体及自噬溶酶体的变化)。结果蛋白免疫印迹法证实siRNA-a及siRNA-b有较好的干扰效果。成功干扰Cofilin-1后,HEK293细胞的p62表达下调、LC3Ⅱ/LC3Ⅰ比值增大、自噬体和自噬溶酶体增多。结论 siRNA能有效干扰Cofilin-1的表达,Cofilin-1参与细胞自噬过程。

Objective To design the small interfering RNA （siRNA） of Cofilin-1, and to explore the effect of Cofilin-1 siRNAs on autophagy.Methods Three pairs of siRNA targeting human Cofilin-1 were designed using online tools and according to the reported literatures.The protein expression of Cofilin-1, p62, LC3Ⅰ and LC3Ⅱ were detected by Western blot before and after HEK293 cells were transfected by Cofilin-1 siRNA.Autophagy flux （the changes of autophgosome and autolysosome） was observed by mRFP-GFP-LC3 double fluorescence.Results The expression of Cofilin-1 was knocked down by siRNA-a and siRNA-b successfully.After down-regulation of Cofilin-1 effectively, the expression of p62 was decreased, and the ratio of LC3Ⅱ/LC3Ⅰ was increased in HEK293 cells.Furthermore, autophgosome and autolysosome were enhanced.Conclusion siRNA can effectively knock down the expression of Cofilin-1;Cofilin-1 is involved in cellular autophagy process.