miR-338-3p通过负性调节cPKCγ表达减轻小鼠神经元缺血损伤  

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

作　　者：魏海萍[1] 郭佳[1] 葛朝明[1] 邵康梅 王浩泰 卢妮 王欢[1] WEI Hai-ping;GUO Jia;GE Zhao-ming;SHAO Kang-mei;WANG Hao-tai;LU Ni;WANG Huan(Department of Neurology,the Second Hospital of Lanzhou University;the Second Clinical Medical College,Lanzhou University,Lanzhou 730030,China)

机构地区：[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

出　　处：《基础医学与临床》2020年第7期917-922,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(81960225);甘肃省自然科学基金(17JR5RA240)。

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cPKCγ3′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);cPKCγ3′UTR存在1个miR-338-3p结合位点,miR-338-3p与cPKCγmRNA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。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 cPKCγprotein 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 cPKCγmRNA;using bioinformatics to detect the binding sites of cPKCγ3′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 cPKCγprotein 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);cPKCγ3′UTR had one miR-338-3p binding site;the miR-338-3p mRNA level showed an opposite trend to that of cPKCγmRNA 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 cPKCγand so to alleviate the injury of ischemic neurons.

关 键 词：miR-338-3p 蛋白激酶CΓ 大脑中动脉阻断 氧糖剥夺 

分 类 号：R743.3[医药卫生—神经病学与精神病学]