miR-155调控线粒体自噬对足细胞损伤的机制研究

Study on the mechanism of miR-155 regulating mitophagy on podocyte injury

目的探讨miR-155对足细胞凋亡的影响,并深入研究其机制。方法MPC5细胞(经H 2O 2预处理)分为miR-155抑表达组、miR-155过表达组、对照(空质粒)组、空白组。分别采用荧光实时定量RT-PCR和Western blot法检测线粒体自噬通路相关因子FOXO1、PINK1、Parkin、LC3Ⅱ、Beclin1、p62及凋亡相关因子Caspase-3、Bax、Bcl-2 mRNA或蛋白表达;JC-1探针检测线粒体膜电位;ELISA检测细胞内ROS含量;CCK8、流式细胞术检测细胞活性及凋亡。结果相较于对照组,miR-155抑表达组的FOXO1、PINK1、Parkin、LC3Ⅱ、Beclin1和Bcl-2表达明显升高,细胞活性和线粒体膜电位明显增加(P<0.05),p62、Caspase-3和Bax的表达明显降低,ROS含量和细胞凋亡率显著下降(P<0.05);miR-155过表达组的FOXO1、PINK1、Parkin、LC3Ⅱ、Beclin1、Bcl-2的表达明显下调,细胞活性和线粒体膜电位明显降低(P<0.05),p62、Caspase-3和Bax表达,细胞内ROS含量和细胞凋亡率显著升高(P<0.05)。对照组与空白组的结果比较无统计学意义(P>0.05)。结论上调miR-155表达,可促进足细胞凋亡,其机制可能与miR-155靶向FOXO1,抑制线粒体自噬,促进足细胞氧化应激损伤有关。

Objective To investigate the effect of miR-155 on podocyte apoptosis and further study its mechanism.Methods MPC5 cells(pretreated with H 2O 2)were divided into miR-155 inhibition group,miR-155 overexpression group,control(empty plasmid)group and blank group.Real-time quantitative RT-PCR and Western blot were used to detect mRNA or protein expressions of mitophagy pathway-related factors(FOXO1,PINK1,Parkin,LC3Ⅱ,Beclin1,p62),apoptosis-related factors(Caspase-3,Bax,Bcl-2mRNA.)or protein expression.JC-1 probe was used to detect mitochondrial membrane potential.ELISA was used to detect intracellular ROS content.CCK8 and flow cytometry were used to detect cell viability and apoptosis.Results Compared with the control group,the expression of FOXO1,PINK1,Parkin,LC3Ⅱ,Beclin1,and Bcl-2 in the miR-155 inhibition group increased significantly,while cell activity and mitochondrial membrane potential increased significantly(P<0.05).The expression of p62,Caspase-3 and Bax decreased significantly,and the content of ROS and apoptotic rate decreased significantly(P<0.05).In addition,the expression of FOXO1,PINK1,Parkin,LC3,Beclin1,and Bcl-2 in the miR-155 overexpression group decreased significantly,cell activity and mitochondrial membrane potential decreased significantly(P<0.05),and the expression of p62,Caspase-3 and Bax increased significantly,the content of ROS and apoptotic rate increased significantly(P<0.05).There was no significant difference in the above-mentioned results between the control group and the blank group(P>0.05).Conclusion Up-regulating miR-155 expression can promote podocyte apoptosis,and the mechanism may be related to mir-155 targeting FOXO1,inhibiting mitochondrial autophagy and promoting oxidative stress injury of podocytes.