摘要
目的探讨微小RNA-486-5p(miR-486-5p)对1-甲基-4-苯基吡啶离子(MPP+)诱导的帕金森病(PD)模型细胞凋亡及自噬的影响。方法用MPP+诱导SK-N-SH细胞建立PD细胞模型,采用实时荧光定量聚合酶链反应(qRT-PCR)与Western印迹平行检测PD细胞模型中miR-486-5p、瞬时受体电位(TRP)M2的表达;将miR-486-5p mimics、miR-NC、si-TRPM2、si-NC转染至SK-N-SH细胞,转染后细胞分为Con组、MPP+组、MPP++miR-486-5p组、MPP++miR-NC组、MPP++si-TRPM2组、MPP++si-NC组。流式细胞仪分析PD细胞模型凋亡率;Western印迹检测PD细胞模型自噬蛋白标记物微管相关蛋白轻链(LC)3Ⅱ、LC3Ⅰ、线粒体功能相关蛋白线粒体融合蛋白(Mfn)2、核呼吸因子(NRF)1及凋亡蛋白B淋巴细胞瘤(Bcl)-2、B淋巴细胞瘤-2相关蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶(cleaved-caspase)3表达情况;双荧光素酶报告基因检测验证miR-486-5p与TRPM2的靶向调控作用。结果MPP+处理后的SK-N-SH细胞凋亡率明显升高(P<0.05),LC3Ⅱ、Bax、cleaved-caspase3蛋白表达水平明显升高(P<0.05),而Mfn2、NRF1、Bcl-2蛋白表达水平明显降低(P<0.05);PD细胞模型中miR-486-5p表达水平降低(P<0.05),TRPM2表达水平升高(P<0.05);PD细胞模型中miR-486-5p过表达与干扰TRPM2的表达可明显降低细胞凋亡率与LC3Ⅱ、Bax、cleaved-caspase3蛋白表达(P<0.05),提高Mfn2、NRF1、Bcl-2蛋白表达(P<0.05);双荧光素酶报告实验证明miR-486-5p可靶向调控TRPM2的表达;TRPM2过表达可逆转miR-486-5p过表达对MPP+诱导的SK-N-SH细胞中线粒体功能相关蛋白、自噬相关蛋白表达及细胞凋亡的作用。结论miR-486-5p过表达可通过下调TRPM2的表达而抑制PD模型细胞凋亡及自噬。
Objective To investigate the effect of microRNA-486-5p(miR-486-5p)on apoptosis and autophagy in Parkinson disease(PD)model induced by 1-methyl-4-phenylpyridinium ion(MPP+).Methods The PD cell model was established by inducing SK-N-SH cells with MPP+,and the expression of miR-486-5p and TRPM2 in PD cell model was detected by qRT-PCR and Western blot.miR-486-5p mimics,miR-NC,si-TRPM2,and si-NC were transfected into SK-N-SH cells.After transfection,the cells were divided into Con group,MPP+group and MPP++miR-486-5p group,MPP++miR-NC group,MPP++si-TRPM2 group,MPP++si-NC group.The apoptosis rate of PD cell model was analyzed by flow cytometry.Western blot was used to detect the expression of autophagic protein markers LC3Ⅱ,LC3Ⅰ,mitochondrial function-related proteins Mfn2,NRF1 and apoptosis proteins Bcl-2,Bax and cleaved-caspase3.Dual luciferase reporter assay was used to validate the targeted regulation of miR-486-5p and TRPM2.Results The apoptosis rate of SK-N-SH cells treated with MPP+was significantly increased(P<0.05),and the expression levels of LC3Ⅱ,Bax and cleaved-caspase3 were significantly increased(P<0.05),while Mfn2,NRF1 and Bcl-2 proteins were significantly decreased(P<0.05).The expression level of miR-486-5p was decreased in PD cell model(P<0.05),and the expression level of TRPM2 was increased(P<0.05).Over-expression of miR-486-5p and interference of TRPM2 expression in PD cell model significantly decreased apoptosis rate and expression of LC3Ⅱ,Bax,cleaved-caspase3 protein(P<0.05),and increased Mfn2,NRF1,Bcl-2 protein expression(P<0.05).The dual luciferase reporter assay demonstrated that miR-486-5p could target the regulation of TRPM2 expression.Over-expression of TRPM2 reversed the effect of miR-486-5p over-expression on MPP+-induced mitochondrial function-related proteins,autophagy-related protein expression,and apoptosis in SK-N-SH cells.Conclusions Over-expression of miR-486-5p could inhibit apoptosis and autophagy in PD model cells by down-regulating the expression of TRPM2.
作者
彭秀华
柳明杰
PENG Xiu-Hua;LIU Ming-Jie(Internal Medicine-Neurology,Jinzhou Central Hospital,Jinzhou 121000,Liaoning,China)
出处
《中国老年学杂志》
CAS
北大核心
2021年第1期115-122,共8页
Chinese Journal of Gerontology
基金
国家自然科学基金委员会资助项目(2014022001)。
作者简介
通信作者:柳明杰(1976-),男,博士,副教授,主要从事神经内科相关疾病研究;第一作者:彭秀华(1976-),女,硕士,副主任护师,主要从事神经内科相关疾病研究。
