摘要
目的探讨微小RNA(miR)-486对1-甲基-4-苯基吡啶离子(MPP^(+))诱导的体外帕金森病(PD)PC12细胞模型凋亡的影响和机制。方法实验分成对照(control)组、PD组(MPP^(+)诱导PC12细胞)、miR-NC组[MPP^(+)诱导PC12细胞转染模拟(mimics)对照(mimics control)]、miR-486组(MPP^(+)诱导PC12细胞转染miR-486 mimics)、miR-486+载体(vector)组(共转染miR-486 mimics和pcDNA3.1)、miR-486+TRIM10组(共转染miR-486 mimics和pcDNA3.1-TRIM10),每组n=9。CCK-8法分析细胞增殖变化,流式细胞术分析细胞凋亡水平变化,Western blotting分析Bax和Bcl-2蛋白表达变化,硫代巴比妥酸法检测细胞中丙二醛(MDA)含量,荧光探针法检测细胞中活性氧簇(ROS)水平,二硝基苯肼分析培养液上清中乳酸脱氢酶(LDH)水平。用生物信息学软件预测miR-486的靶基因,双荧光素酶报告基因实验检测miR-486、TRIM10靶向关系。结果与control组比较,PD组细胞存活率、Bcl-2蛋白表达降低,细胞凋亡率、Bax蛋白表达、MDA、ROS和LDH水平升高。与miR-NC组比,miR-486组细胞存活率、Bcl-2蛋白表达升高,细胞凋亡率、Bax蛋白表达、MDA、ROS和LDH水平降低。MiR-486靶向下调TRIM10表达。与miR-486+vector组比较,miR-486+TRIM10组细胞存活率、Bcl-2蛋白水平降低,细胞凋亡率、Bax蛋白水平、MDA、ROS和LDH水平升高。结论上调miR-486可通过靶向抑制TRIM10减少MPP^(+)诱导的体外帕金森病PC12细胞模型的凋亡。
Objective To study the effect and mechanism of microRNA-486(miR-486)on 1-methyl-4-phenylpyridine(MPP^(+))-induced apoptosis of Parkinson’s disease(PD)PC12 cells in vitro.Methods The experiment was divided into control group,PD(MPP^(+)induced PC12 cells),miR-NC(transfected mimics control,MPP^(+)induced PC12 cells),miR-486 group(transfected miR-486 mimics,MPP^(+)induced PC12 cells),miR-486+vector group(co-transfected miR-486 mimics,pcDNA3.1),miR-486+TRIM10 group(co-transfected miR-486 mimics,pcDNA3.1-TRIM10),n=9 each group.CCK-8 method was used to analyze cell proliferation changes,flow cytometry was used to analyze cell apoptosis levels,Western blotting was used to analyze changes in Bax and Bcl-2 protein expression,thiobarbituric acid method was used to detect malondialdehyde(MDA)content in cells,fluorescence probe method was used to detect the level of reactive oxygen species(ROS)in the cells,and 2,4-dinitrophenylhydrazine was used to analyze the level of lactate dehydrogenase(LDH)in the culture supernatant.Bioinformatics software was used to predict the target genes of miR-486,and the detection of targeting relationship between imiR-486 and TRIM10 by dual luciferase reporter gene assay.Results Compared with the control group,the cell survival rate and Bcl-2 protein expression in the PD group decreased,while the apoptosis rate,Bax protein expression,MDA,ROS and LDH levels increased.Compared with the miR-NC group,the cell survival rate and Bcl-2 protein expression in the miR-486 group were increased,and the apoptosis rate,Bax protein expression,MDA,ROS and LDH levels were decreased.MiR-486 targeted down-regulation of TRIM10 expression.Compared with the miR-486+vector group,the miR-486+TRIM10 group decreased the cell survival rate and Bcl-2 protein level,while the apoptosis rate,Bax protein level,MDA,ROS and LDH levels increased.Conclusion Up-regulation of miR-486 targeted and inhibited TRIM10 to reduce MPP^(+)-induced apoptosis in in vitro Parkinson’s PC12 cell models.
作者
廖建云
江新柳
谢为法
LIAO Jian-yun;JIANG Xin-liu;XIE Wei-fa(Department of Neurology,Jingdezhen Second People’s Hospital,Jiangxi Jingdezhen 333000,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2022年第4期424-431,共8页
Acta Anatomica Sinica
基金
江西省卫生计生委科研课题项目(20187185)。
