miR-7和miR-153抑制α-syn表达在百草枯诱导多巴胺能神经元损伤中的作用机制  

The mechanism of miR-7 and miR-153 inhibitingα-SYN expression in paraquat induced dopaminergic neuronal injury

作　　者：王一帆[1] 田甜 闫伟广 王楷栋 张宝馥 张冰洋 黄敏[1] WANG Yi-fan;TIAN Tian;YAN Wei-guang;WANG Kai-dong;ZHANG Bao-fu;ZHANG Bing-yang;HUANG Min(Ningxia Medical University,Yinchuan Ningxia 750004,China)

机构地区：[1]宁夏医科大学,宁夏银川750004

出　　处：《毒理学杂志》2021年第2期152-158,共7页Journal of Toxicology

基　　金：宁夏自然科学基金重点项目(2020AAC02018)。

摘　　要：目的探讨miRNA-7和miRNA-153在百草枯(PQ)诱导多巴胺能神经元损伤中抑制α-syn表达的作用机制。方法运用TargetScan和HMDD等基因预测软件预测miR-7和miR-153与α-synuclein蛋白编码基因(SNCA)结合位点及与帕金森病相关性。选择高表达内源性α-syn的HEK293T细胞作为转染细胞株,采用化学合成miRNAs模拟物和抑制剂转染HEK293T细胞调控其miR-7和miR-153表达,蛋白免疫印迹法(Western blot)和实时荧光定量PCR(RT-qPCR)检测细胞α-syn基因与蛋白表达水平,观察miR-7和miR-153与内源性α-syn基因表达的关系;为了进一步研究在环境化学物PQ诱导下miR-7和miR-153对α-syn基因的调控作用,以人神经母细胞瘤细胞(SH-SY5Y)作为多巴胺能神经元体外模型,不同浓度PQ(0、25、50、100、200、400和800μmol/L)处理细胞24 h,100μmol/L PQ处理细胞不同时间(0、12、24、36、48、60和72 h),CCK8法检测细胞增殖活性;RT-qPCR检测miR-7、miR-153表达,Western blot检测α-syn蛋白表达水平;对多巴胺能神经元分别转染miRNA模拟物和miRNA抑制剂后再进行PQ诱导24 h,Western blot和RT-qPCR检测α-syn蛋白及基因表达水平。结果生物信息学分析显示,α-syn编码基因(SNCA)3′-UTR有两个片段能够与miR-7(序列120~127)和miR-153(序列459~465)结合;上调HEK293T细胞miR-7和miR-153表达后,α-syn基因和蛋白表达水平均明显下降,尤其在100 nmol/L浓度下降明显(P<0.05);相反,抑制miRNA表达后α-syn基因和蛋白表达水平升高(P<0.05),说明miR-7和miR-153能够靶向下调α-syn基因进而抑制α-syn蛋白表达。进一步使用外源化学物PQ处理多巴胺能神经元,染毒组细胞增殖活性随剂量和时间的增加而下降(P<0.05);细胞内α-syn蛋白表达水平升高(P<0.05);而miR-7和miR-153表达水平明显降低(P<0.05);向细胞转染miRNA模拟物以上调miRNA表达后,α-syn mRNA和蛋白表达均被抑制(P<0.05),而转染miRNA抑制剂抑制miRNA表达,α-syn mRNA及蛋白表达增加(P<0.0Objective To investigate the protective role of miR-7 and miR-153 in paraquat(PQ)-induced dopaminergic neuron injury.Methods TargetScan and HMDD were used to predict the binding sites of miR-7 and miR-153 withα-synuclein gene SNCA and the correlation with Parkinson′s disease.HEK293 T cells with high expression of endogenousα-syn were selected as transfected cell lines,and miRNA mimics and miRNA inhibitors of different concentrations were transfected.Western blot and real-time fluorescence quantitative PCR(RT-qPCR)were used to detect the expression levels ofα-syn protein and mRNA respectively.The relative survival rate of cells was measured by CCK8.Human neuroblastoma SH-SY5 Y cells were used as in vitro models of dopaminergic neurons.Cells were treated with PQ(0,25,50,100,200,400,800μmol/L)for 24 h,and PQ(100μmol/L)for different times(0,12,24,36,48,60,72 h).CCK8 was used to detect the relative survival rate of cells.Expression changes of miR-7 and miR-153 were detected by RT-qPCR,and expression levels ofα-syn protein were detected by Western blot.After transfection of miRNA mimics into dopaminergic neuron,the cells were induced by PQ for 24 h,and expression levels ofα-syn protein and mRNA were respectively detected by Western blot and RT-qPCR.Results Bioinformatics analysis showed that two fragments ofα-syn mRNA 3′-UTR were binding to miR-7(located in sequence 120-127)and miR-153(sequence 459-465),respectively,and the two miRNAs were able to down-regulate SNCA in the Parkinson′s disease associated network diagram.When miRNA expression in HEK293 T cells was promoted,the relative survival rate of the cells was increased(P<0.05),and the expression levels ofα-syn mRNA and protein were significantly decreased(P<0.05),especially at 100 nmol/L concentration(P<0.05);In contrast,after miRNA expression was inhibited,α-syn mRNA and protein expression levels increased(P<0.05);Dopaminergic neuron were treated with PQ at different concentrations.Compared with the control group,the proliferation activity of dopa

关 键 词：MiR-7 MiR-153 Α-突触核蛋白 多巴胺能神经元 百草枯 

分 类 号：R114[医药卫生—卫生毒理学] R99[医药卫生—公共卫生与预防医学]