miR-24对过氧化氢诱导的HLE-B3细胞凋亡的影响及其与线粒体Smac/Diablo-XIAP-caspase-9/3凋亡途径的关系  

Effect of miR-24 on hydrogen peroxide-induced apoptosis of HLE-B3 cells and its correlation with Smac/Diablo-XIAP-caspase-9/3 pathway

作　　者：闫利霞 黄晓 张鑫 郑广瑛[3] YAN Lixia;HUANG Xiao;ZHANG Xin;ZHENG Guangying(Children’s Ophthalmology of Kaifeng Central Hospital,Kaifeng 475000,Henan Province,China;Children’s Ophthalmology,Kaifeng Eye Hospital,Kaifeng 475000,Henan Province,China;Department of Ophthalmology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450002,Henan Province,China)

机构地区：[1]开封市中心医院小儿眼科,河南省开封市475000 [2]开封眼病医院小儿眼科,河南省开封市475000 [3]郑州大学第一附属医院眼科,河南省郑州市450002

出　　处：《眼科新进展》2021年第5期417-421,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金面上项目(编号:81670836)。

摘　　要：目的探讨微小RNA\|24(miR-24)对过氧化氢(H_(2)O_(2))诱导的HLE-B3细胞凋亡的影响,分析其与线粒体中第二个线粒体衍生的胱天蛋白酶激活因子/低等电位点的凋亡抑制蛋白的直接结合蛋白(the second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI,Smac/Diablo)-XIAP-caspase-9/3凋亡途径的关系。方法将HLE-B3细胞分为对照组、H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组。先按照miR-24抑制剂、miR-24 NC试剂盒说明书转染H_(2)O_(2)+miR-24抑制剂组和H_(2)O_(2)+miR-24 NC组细胞24 h,然后向H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组HLE-B3细胞中加入200μmol·L^(-1)H_(2)O_(2)干预24 h,收集细胞用于后续实验;将正常培养的HLE-B3细胞设置为对照组。采用实时荧光定量PCR检测各组HLE-B3细胞miR-24表达情况,CCK-8法检测各组HLE-B3细胞生长情况,用流式细胞仪检测各组细胞凋亡情况,计算各组细胞存活率和细胞凋亡率。采用荧光探针JC-1法检测线粒体膜电位变化情况(利用红绿荧光强度比反映线粒体膜电位);采用蛋白免疫印迹法检测各组HLE-B3细胞线粒体和细胞质分级中Smac/Diablo及细胞质分级中XIAP、caspase-9和caspase-3蛋白表达情况。对所得数据进行统计学分析。结果当H_(2)O_(2)浓度大于200μmol·L^(-1)时,细胞存活率均小于50%,本实验以200μmol·L^(-1)作为最适H_(2)O_(2)浓度。对照组、H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组细胞miR-24相对表达量分别为1.04±0.02、2.73±0.09、2.69±0.07和1.15±0.06;与对照组相比,H_(2)O_(2)组细胞中miR-24表达升高(P<0.05);与H_(2)O_(2)组和H_(2)O_(2)+miR-24 NC组相比,H_(2)O_(2)+miR-24抑制剂组中miR-24表达降低(均为P<0.05),但H_(2)O_(2)组与H_(2)O_(2)+miR-24 NC组中miR-24表达差异无统计学意义(P>0.05)。与H_(2)O_(2)组和H_(2)O_(2)+miR-24 NC组相比,H_(2)O_(2)+miR-24抑制剂组细胞存活率显著�Objective To explore the effect of microRNA-24(miR-24)on apoptosis of human lens epithelial cells(HLE-B3)induced by hydrogen peroxide(H_(2)O_(2)),and to analyze its relationship with mitochondrial Smac/Diablo-XIAP-caspase-9/3 apoptosis pathway.Methods HLE-B3 cells were divided into control group,H_(2)O_(2)group,H_(2)O_(2)+miR-24 NC group and H_(2)O_(2)+miR-24 inhibitor group.Cells in H_(2)O_(2)+miR-24 inhibitor group and H_(2)O_(2)+miR-24 NC group were transfected for 24 h according to the miR-24 inhibitor and miR-24 NC kit instructions,and then 200μmol·L^(-1)H_(2)O_(2)was added to the cells in the H_(2)O_(2)group,H_(2)O_(2)+miR-24 NC group and H_(2)O_(2)+miR-24 inhibitor group for 24 h,and the cells were collected for subsequent experiments.The normally cultured HLE-B3 cells were set as the control group.Real-time fluorescent quantitative PCR method was used to detect the expression of miR-24 in HLE-B3 cells;CCK-8 method was used to detect the growth of HLE-B3 cells in each group;flow cytometry was used to detect the apoptosis of HLE-B3 cells,and finally,cell survival rate and cell apoptosis rate were calculated.Fluorescent probe JC-1 method was used to detect the change of mitochondrial membrane potential(The ratio of red-green fluorescence intensity can reflects the mitochondrial membrane potential).Western blot was used to detect the expression of the second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI(Smac/Diablo),XIAP,caspase-9 and caspase-3 proteins.Statistical analysis was performed on the obtained data.Results When the H_(2)O_(2)concentration was greater than 200μmol·L^(-1),the cell survival rate was less than 50%,so in this experiment,200μmol·L^(-1)was used as the optimal H_(2)O_(2)concentration.The relative expression level of miR-24 in the control group,H_(2)O_(2)group,H_(2)O_(2)+miR-24 NC group and H_(2)O_(2)+miR-24 inhibitor group was 1.04±0.02,2.73±0.09,2.69±0.07 and 1.15±0.06,respectively;compared with control group,the expression level of miR-24 in H_

关 键 词：微小RNA-24 过氧化氢 人晶状体上皮细胞 胱天蛋白酶激活因子 低等电位点的凋亡抑制蛋白 细胞凋亡 

分 类 号：R776[医药卫生—眼科]