小鼠神经细胞氧糖剥夺-复糖复氧时miR-93-5p与线粒体融合蛋白2的关系  

Relationship between microRNA-93-5p and mitochondrial fusion protein-2 in mouse nerve cells subjected to oxygen-glucose deprivation and reoxygenation

作　　者：王炳琪 刘同帅 于群 陈怀龙[2] 时飞[2] Wang Bingqi;Liu Tongshuai;Yu Qun;Chen Huailong;Shi Fei(Department of Anesthesiology,Weifang Medical College,Weifang 261053,China;Department of Anesthesiology,Qingdao Municipal Hospital Affiliated to Qingdao University,Qingdao 266071,Chin)

机构地区：[1]潍坊医学院麻醉学院,潍坊261053 [2]青岛大学附属青岛市市立医院麻醉科,青岛266071

出　　处：《中华麻醉学杂志》2022年第5期611-615,共5页Chinese Journal of Anesthesiology

基　　金：青岛市民生科技计划项目(19-6-1-50-nsh)。

摘　　要：目的评价小鼠神经细胞氧糖剥夺-复糖复氧时微小RNA-93-5p(miR-93-5p)与线粒体融合蛋白2(Mfn2)的关系。方法体外培养小鼠神经母细胞瘤细胞至对数生长期。实验Ⅰ采用随机数字表法将细胞分为5组(n=20):对照组(C组)、氧糖剥夺-复糖复氧组(OGD/R组)、miR-93-5p抑制剂组(I组)、siRNA-Mfn2+miR-93-5p抑制剂组(siMfn2+I组)和miR-93-5p阴性对照组(NC组)。氧糖剥夺:在低糖平衡盐溶液、37℃5%CO_(2)-95%N2的环境中培养3 h,复糖复氧:在正常培养基、37℃5%CO_(2)-95%空气中复氧24 h。I组、siMfn2+I组与NC组在模型制备前48 h分别转染miR-93-5p抑制剂、miR-93-5p抑制剂+siRNA-Mfn2及阴性对照miRNA。采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡率,qRT-PCR法检测miR-93-5p、Mfn2 mRNA表达水平,Western blot法检测Mfn2表达水平。实验Ⅱ构建野生型(WT)-Mfn2和突变型(MUT)-Mfn2质粒,并分别与miR-93-5p模拟物和miR-93-5p空白对照(miR-93-5p NC)载体转染至神经母细胞瘤细胞,转染48 h后,采用随机数字表法将细胞分为4组(n=5):miR-93-5p NC-WT-Mfn2共转染组、miR-93-5p模拟物-WT-Mfn2共转染组、miR-93-5p NC-MUT-Mfn2共转染组和miR-93-5p模拟物-MUT-Mfn2共转染组。采用双荧光素酶实验检测荧光素酶活性。结果实验Ⅰ与C组比较,其余各组细胞活力降低,细胞凋亡率升高,miR-93-5p表达上调,Mfn2及其mRNA表达下调(P<0.05);与OGD/R组或NC组比较,I组细胞活力升高,细胞凋亡率降低,miR-93-5p表达下调,Mfn2及其mRNA表达上调(P<0.05);与I组比较,siMfn2+I组细胞活力降低,细胞凋亡率升高,Mfn2及其mRNA表达下调(P<0.05)。实验Ⅱ与miR-93-5p NC-WT-Mfn2共转染组比较,miR-93-5p模拟物-WT-Mfn2共转染组荧光素酶活性降低(P<0.05);与miR-93-5p NC-MUT-Mfn2共转染组比较,miR-93-5p模拟物-MUT-Mfn2共转染组荧光素酶活性差异无统计学意义(P>0.05)。结论miR-93-5p表达上调,进而靶向下调Mfn2表达,可能是小鼠神经细胞氧糖剥夺-复糖复�Objective To evaluate the relationship between microRNA-93-5p and mitochondrial fusion protein-2(Mfn2)in mouse nerve cells subjected to oxygen-glucose deprivation and reoxygenation(OGD/R).Methods Mouse neuroblastoma cells were cultured in vitro to logarithmic growth phase.ExperimentⅠCells were divided into 5 groups(n=20 each)by the random number table method:control group(group C),group OGD/R,miR-93-5p inhibitor group(group I),siRNA-mfn2 plus miR-93-5p group(group siMfn2+I)and miR-93-5p negative control group(group NC).Oxygen-glucose deprivation:the cells were cultured for 3 h in a low-glucose balanced salt solution at 37℃in an environment of 5%CO_(2)-95%N2.Restoration of oxygen and glucose:the cells were cultured in normal medium at 37℃in 5%CO_(2)-95%air for 24 h.Group I,group siMfn2+I and group NC were transfected with miR-93-5p inhibitor,miR-93-5p inhibitor plus siRNA-mfn2 and negative control miRNA,respectively,at 48 h before the OGD/R model was developed.Cell viability was measured by CCK-8 assay.Cell apoptosis rate was measured by flow cytometry.Quantitative real-time polymerase chain reaction was used to detect the expression of miR-93-5p and Mfn2 mRNA.Western blot was used to detect Mfn2 protein expression.ExperimentⅡThe wild-type(WT)-Mfn2 and mutant(MUT)-Mfn2 were constructed and transfected into neuroblastoma cells with miR-93-5p mimic and miR-93-5p blank control(miR-93-5pNC),respectively.The cells were divided into 4 groups(n=5 each)after 48 h of transfection by the random number table method:miR-93-5p NC-WT-Mfn2 co-transfection group,miR-93-5p mimic-WT-Mfn2 co-transfection group,miR-93-5p NC-MUT-Mfn2 co-transfection group,and miR-93-5p mimic-MUT-Mfn2 co-transfection group.The activity of luciferases was measured by double luciferase assay.Results ExperimentⅠCompared with group C,the cell viability was significantly decreased,the apoptosis rate of cells was increased,the expression of miR-93-5p was up-regulated,and the expression of Mfn2 protein and mRNA was down-regulated in the other gorups

关 键 词：微RNAS 线粒体蛋白质类 再灌注损伤 脑 

分 类 号：R614[医药卫生—麻醉学]