miR-338-3p通过负性调节cPKCγ表达减轻小鼠神经元缺血损伤

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (7): 917-922.

miR-338-3p通过负性调节cPKCγ表达减轻小鼠神经元缺血损伤

魏海萍¹, 郭佳¹, 葛朝明¹, 邵康梅², 王浩泰², 卢妮², 王欢^1*

1.兰州大学第二医院神经内科,甘肃兰州 730030;
2.兰州大学第二临床医学院,甘肃兰州 730030

收稿日期:2020-03-18 修回日期:2020-03-24 出版日期:2020-07-05 发布日期:2020-06-29
通讯作者: ^*sjnk11@sina.com
基金资助:
国家自然科学基金(81960225); 甘肃省自然科学基金(17JR5RA240)

miR-338-3p alleviates neuronal ischemic injury of mice through negatively regulating cPKCγ expression

WEI Hai-ping¹, GUO Jia¹, GE Zhao-ming¹, SHAO Kang-mei², WANG Hao-tai², LU Ni², WANG Huan^1*

1. Department of Neurology, the Second Hospital of Lanzhou University;
2. the Second Clinical Medical College, Lanzhou University, Lanzhou 730030, China

Received:2020-03-18 Revised:2020-03-24 Online:2020-07-05 Published:2020-06-29
Contact: ^*sjnk11@sina.com

摘要/Abstract

摘要： 目的评估miR-338-3p对氧糖剥夺(OGD)/复糖复氧(R)神经元损伤的影响及其机制。方法建立小鼠脑中动脉阻塞(MCAO)和神经元氧糖剥夺(OGD)模型;Western blot检测1 h MCAO/R 24、48和72 h小鼠脑梗死区周围皮质(n=5)和1 h OGD/R 0、6、12和24 h神经元(n=5)cPKCγ蛋白表达量;实时定量PCR检测cPKCγ及miR-338-3p mRNA水平;生物信息学分析cPKCγ 3′UTR与miR-338-3p结合位点;噻唑兰(MTT)比色法和乳酸脱氢酶(LDH)法测定神经元(n=6)的存活率和凋亡率。采用免疫荧光与高内涵细胞成像分析技术(HCA)检测caspase-3蛋白表达情况。结果 1 h MCAO/R 24、48和72 h小鼠脑梗死区周围皮层和1 h OGD/R 0、6、12和24 h 神经元中cPKCγ蛋白及mRNA水平均明显上升(P＜0.001);cPKCγ 3′UTR存在1个miR-338-3p结合位点,miR-338-3p与cPKCγ mRNA水平呈相反趋势;过表达miR-338-3p后1 h OGD/R 24 h神经元存活率下降(P＜0.001),调亡率上升(P＜0.001),caspase-3蛋白平均荧光强度值增高(P＜0.001),抑制miR-338-3p后1 h OGD/R 24 h神经元存活率上升(P＜0.001),调亡率下降(P＜0.001),caspase-3蛋白平均荧光强度值降低(P＜0.001)。结论 miR-338-3p通过负性调节cPKCγ表达而使神经元存活率上升,凋亡率下降,减轻缺血神经元损伤。

关键词: miR-338-3p, 蛋白激酶Cγ, 大脑中动脉阻断, 氧糖剥夺

Abstract: Objective To investigate the effect of miR-338-3p on ischemic injury of neurons after oxgen glucose deprivation(OGD)/reoxgenation(R). Methods In vivo model of middle cerebral artery occlusion(MCAO) was developed in mice and in vitro model of oxygen glucose deprivation(OGD) was developed with in cortical neurons. Using Western blot to detect cPKCγ protein levels in peri-infarct cortex of mice(n=5) with 1 h MCAO/R 24,48 and 72 h and in neurons(n=5) with 1 h OGD/R 0,6,12 and 24 h; using quantitative RT-qPCR to detect miR-338-3p and cPKCγ mRNA; using bioinformatics to detect the binding sites of cPKCγ 3′UTR and miR-338-3P. Using MTT and LDH methods to detect the survival rate and apoptosis rate of nurons(n=6); using immunofluorescence and HCA to detect the expression of caspase-3 in neurons(n=5). Results cPKCγ protein and mRNA significantly increased in mice with 1 h MCAO/R 24,48 and 72h and in neurons with 1 h OGD/R 0,6,12 and 24 h(P＜0.001); cPKCγ 3′UTR had one miR-338-3p binding site; the miR-338-3p mRNA level showed an opposite trend to that of cPKCγ mRNA level; after over-expression miR-338-3p, the survival rate decreased(P＜0.001) and the apoptosis rate increased(P＜0.001) and the mean fluorescence intensity of caspase-3 increased(P＜0.001) in neurons after 1 h OGD/R 24 h. After inhibition miR-338-3p, the survival rate increased(P＜0.001), the apoptosis rate decreased(P＜0.001) and the mean fluorescence intensity of caspase-3 decreased(P＜0.001) in neurons after 1 h OGD/R 24 h. Conclusions miR-338-3p enhances the survival rate and reduces apoptosis rate of ischemic neurons by negatively regulation of cPKCγ and so to alleviate the injury of ischemic neurons.

Key words: miR-338-3p, cPKCγ, middle cerebral artery occlusion(MCAO), oxygen glucose deprivation(OGD)

中图分类号:

R743.3

魏海萍, 郭佳, 葛朝明, 邵康梅, 王浩泰, 卢妮, 王欢. miR-338-3p通过负性调节cPKCγ表达减轻小鼠神经元缺血损伤[J]. 基础医学与临床, 2020, 40(7): 917-922.

WEI Hai-ping, GUO Jia, GE Zhao-ming, SHAO Kang-mei, WANG Hao-tai, LU Ni, WANG Huan. miR-338-3p alleviates neuronal ischemic injury of mice through negatively regulating cPKCγ expression[J]. Basic & Clinical Medicine, 2020, 40(7): 917-922.

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