微小RNA-486靶向TRIM10抑制帕金森病细胞模型损伤

doi:10.16098/j.issn.0529-1356.2022.04.004

摘要/Abstract

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。

关键词: 微小RNA-486, TRIM10, 帕金森病, 免疫印迹法, PC12细胞

Abstract:

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 TRIM 10 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.

Key words: MicroRNA-486, TRIM10, Parkinson’s disease, Western blotting, PC12 cell

中图分类号:

R741.02

廖建云江新柳谢为法. 微小RNA-486靶向TRIM10抑制帕金森病细胞模型损伤[J]. 解剖学报, 2022, 53(4): 424-431.

LIAO Jian-yun JIANG Xin-liu XIE Wei-fa. MicroRNA-486 targeting TRIM10 to inhibit Parkinson’s disease cell model damage[J]. Acta Anatomica Sinica, 2022, 53(4): 424-431.

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