长链非编码RNA-心肌梗死相关转录本对过氧化氢诱导的人晶状体上皮细胞凋亡和氧化应激的调控作用

Regulation of lncRNA-MIAT on the apoptosis and oxidative stress in H2O2-induced human lens epithelial cells

目的探讨长链非编码RNA(lncRNA)-心肌梗死相关转录本(myocardial infarction-associated transcript,MIAT)对过氧化氢(H_(2)O_(2))诱导的人晶状体上皮细胞凋亡和氧化应激的调控作用。方法体外培养人晶状体上皮细胞株HLE-B3,分为空白对照组、H_(2)O_(2)组、阴性对照组(转染空质粒)、si-MIAT组(慢病毒感染siMIAT质粒)、si-MIAT+miR-22 inhibitor组(慢病毒感染siMIAT质粒+脂质体转染miR-22 inhibitor),除空白对照组外,各组细胞均用100μmol·L^(-1) H_(2)O_(2)培养12 h。实时荧光定量PCR(RT-qPCR)测定各组细胞中lncRNA-MIAT的相对表达水平。采用CCK-8法检测细胞存活率和凋亡率。流式细胞仪检测细胞内活性氧簇(ROS)水平。双荧光素酶和RNA结合蛋白免疫共沉淀实验分析lncRNA-MIAT和miR-22以及p38丝裂原活化蛋白激酶(p38 MAPK)的靶向关系。Western blot检测各组细胞中p38 MAPK、p-p38 MAPK、c-Jun氨基末端激酶(JNK)、p-JNK蛋白表达。结果与空白对照组相比,H_(2)O_(2)组可诱导HLE-B3细胞lncRNA-MIAT表达上调,miR-22表达降低(均为P<0.05)。与空白对照组和阴性对照组相比,si-MIAT组细胞miR-22相对表达量升高,而与si-MIAT组相比,si-MIAT+miR-22 inhibitor组细胞miR-22相对表达量降低,差异均有统计学意义(均为P<0.05)。与H_(2)O_(2)组相比,si-MIAT+H_(2)O_(2)组细胞存活率升高,细胞凋亡率和MDA、ROS含量降低,BAX蛋白、p-p38 MAPK/p38 MAPK比值、p-JNK/JNK比值均降低(均为P<0.05)。经双荧光素酶报告基因分析,lncRNA-MIAT靶向结合miR-22,且p38是miR-22的下游靶基因。与si-MIAT+H_(2)O_(2)组相比,si-MIAT+miR-22 inhibitor+H_(2)O_(2)组细胞存活率降低,细胞凋亡率和MDA、ROS含量增加,BAX蛋白、p-p38 MAPK/p38 MAPK比值、p-JNK/JNK比值均增加(均为P<0.05)。结论lncRNA-MIAT具有海绵吸附miR-22的作用,敲低lncRNA-MIAT可通过上调miR-22表达进而抑制p38 MAPK/JNK通路活化,抑制H_(2)O_(2)诱导的人晶状体上皮细胞凋亡和氧化应激反应。

Objective To investigate the regulation and mechanism of long-strand non-coding RNA-myocardial infarction-related transcription(lncRNA-MIAT)on apoptosis and oxidative stress induced by H_(2)O_(2) in human lens epithelial cells.Methods Human lens epithelial cell line HLE-B3 was cultured in vitro,which were divided into control group,H_(2)O_(2) group,negative control(NC)group,in which cells were transfected with empty plasmid,si-MIAT group,in which cells were transfected with si-MIAT,si-MIAT+miR-22 inhibitor group,in which cells were transfected with siMIAT and miR-22 inhibitor.The cells in H_(2)O_(2) group,NC group,si-MIAT group,si-MIAT+miR-22 inhibitor group were cultured for 12 hours with 100μmol·L^(-1) H_(2)O_(2).The relative expression level of lncRNA-MIAT in groups was determined by RT-qPCR.The cell survival rate and apoptosis rate were detected by CCK-8 and flow cytometry method.Intracellular reactive oxygen species(ROS)levels were measured by fluorescence method.Double Luciferase and RNA immunoprecipitation assay was used to analyze lncRNA-MIAT,miR-22 and p38 mitogen-activated protein kinase(p38 MAPK)targeting.Western blot was used to detect the expression levels of p38 MAPK,p-p38 MAPK,c-Jun N-terminal kinase(JNK)and p-JNK proteins.Results When compared with control group,H_(2)O_(2) induced the up-regulation of lncRNA-MIAT expression in HLE-B3 cells,while miR-22 expression was down-regulated(P<0.05).When compared with control group and NC group,miR-22 expression was up-regulated in si-MIAT group,while miR-22 expression was down-regulated in si-MIAT+miR-22 inhibitor group compared with si-MIAT group,with significant differences(both P<0.05).Compared with H_(2)O_(2) group,the survival rate of cells in si-MIAT+H_(2)O_(2) group increased,while the apoptosis rate and ROS and MDA contents decreased,and the expression levels of BAX protein,the ratio of p-p38 MAPX/p38MAPK,the ratio of p-JNK/JNK also decreased(all P<0.05).By double luciferase reporter gene analysis,lncRNA MIAT targeted miR-22,while p38ɑwas the downstream target gene of miR-22.Compared with si-MIAT+H_(2)O_(2) group,the cell survival rate in si-MIAT+miR-22 inhibitor+H_(2)O_(2) group decreased,and the apoptosis rate and ROS content increased correspondingly,and p-p38 MAPK/p38 MAPK protein ratio and p-JNK/JNK protein ratio also increased significantly(all P<0.05).Conclusion lncRNA-MIAT has the effect of sponge adsorbing miR-22.Knock-down of lncRNA MIAT can inhibit the activation of p38/JNK pathway by up-regulating miR-22 expression,thus saving the apoptosis and oxidative stress response of lens epithelial cells induced by H_(2)O_(2).