siRNA-KCNQ1OT1对人LECs凋亡的抑制作用及其靶向miR-199a-5p机制  

Inhibitory effect of siRNA-KCNQ1OT1 on human lens epithelial cell apoptosis by targeting miR-199a-5p

作　　者：鲁诚[1] 张凤妍[2] 张宇航 张佳娟[1] Lu Cheng;Zhang Fengyan;Zhang Yuhang;Zhang Jiajuan(Department of Ophthalmology,Xinxiang First People's Hospital,Xinxiang 453000;Department of Ophthalmology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]新乡市第一人民医院眼科,新乡453000 [2]郑州大学第一附属医院眼科,郑州450000

出　　处：《中华实验眼科杂志》2022年第6期514-523,共10页Chinese Journal Of Experimental Ophthalmology

摘　　要：目的探讨长链非编码RNA KCNQ1重叠转录物1(KCNQ1OT1)对人晶状体上皮细胞(LECs)凋亡的影响及其机制。方法收集2018年12月至2019年8月在新乡市第一人民医院行白内障手术的23例年龄相关性白内障患者的晶状体前囊膜组织,同时收集20例正常供体眼晶状体前囊膜组织。采用实时荧光定量PCR法检测并比较不同人群晶状体前囊膜组织中KCNQ1OT1和miR-199a-5p mRNA表达水平。体外培养人LECs(SRA01/04),将细胞分为空白对照组、模型对照组、小干扰RNA-阴性对照(siR-NC)组、siR-KCNQ1OT1组、miR-NC组、miR-199a-5p组、siR-KCNQ1OT1+anti-miR-NC组和siR-KCNQ1OT1+anti-miR-199a-5p组,其中空白对照组不做任何处理,模型对照组采用100μmol/L过氧化氢(H_(2)O_(2))培养细胞24 h制作氧化应激损伤模型,其余各组分别采用相应转染试剂按照脂质体法转染6 h后换用100%μmol/L H_(2)O_(2)处理细胞24 h。采用实时荧光定量PCR法检测各组晶状体前囊膜组织和各组LECs细胞中KCNQ1OT1和miR-199a-5p表达水平;采用MTT法检测各组细胞活力值;采用流式细胞术检测各组细胞凋亡率;采用Western blot法检测各组B细胞淋巴瘤/白血病-2(bcl-2)和bcl-2相关X蛋白(bax)表达水平;采用酶联免疫吸附测定(ELISA)法检测各组细胞中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;采用双荧光素酶报告实验检测KCNQ1OT1和miR-199a-5p的关系。结果年龄相关性白内障患者前囊膜内KCNQ1OT1相对表达量为2.41±0.42,明显高于正常人的0.97±0.19,差异有统计学意义(t=14.112,P<0.001);miR-199a-5p相对表达量为0.36±0.12,明显低于正常人的1.04±0.15,差异有统计学意义(t=16.507,P<0.001)。与空白对照组比较,模型对照组中KCNQ1OT1和bax蛋白相对表达量、细胞凋亡率、MDA含量明显增加,miR-199a-5p和bcl-2蛋白相对表达量、细胞活力值、SOD活性值明显降低,差异均有统计学意义(均P<0.001);与siR-NC组比较,siR-KCNQ1OT1组细胞中KCNQ1OT1和baObjective To explore the inhibitory effect of long non-coding RNA(lncRNA)KCNQ1 overlapping transcript 1(KCNQ1OT1)by targeting microRNA-199a-5p(miR-199a-5p)on the apoptosis of human lens epithelial cells(LECs).Methods The anterior lens capsule tissue of 23 age-related cataract patients who underwent cataract surgery in Xinxiang First People's Hospital from December 2018 to August 2019 was collected.At the same time,anterior lens capsules from 20 healthy donor were collected.The expressions of KCNQ1OT1 and miR-199a-5p in the tissues were detected by real-time fluorescence PCR.Human LECs SRA01/04 cultured in vitro were divided into blank control group,model control group,small interfering RNA-negative control(siR-NC)group,siR-KCNQ1OT1 group,miR-NC group,miR-199a-5p group,siR-KCNQ1OT1+anti-miR-NC group and siR-KCNQ1OT1+anti-miR-199a-5p group.No intervention was administered to blank control group.Cells in model control group were cultured with 100μmol/LH_(2)O_(2) for 24 hours to establish oxidative stress injured model,and cells in the other six groups were transfected with corresponding transfection reagents for 6 hours by liposome method according to grouping,and then treated with 100μmol/L H_(2)O_(2) for 24 hours.The expressions of KCNQ1OT1 and miR-199a-5p in lens anterior capsule tissue and LECs cells were determined by real-time fluorescent quantitative PCR.Cell viability was detected with thiazolyl blue(MTT).Cell apoptosis was analyzed by flow cytometry.The expressions of B-cell lymphoma/leukemia-2(bcl-2)and bcl-2 related X protein(Bax)proteins were assayed by Western blot.The superoxide dismutase(SOD)activity and malondialdehyde(MDA)content were measured by enzyme-linked immunosorbent assay(ELISA).The targeting relationship between KCNQ1OT1 and miR-199a-5p was verified by dual luciferase reporter experiment.The study protocol was approved by an Ethics Committee of Xinxiang First People's Hospital(No.2019-001).Written informed consent was obtained from relatives of patient.Results The relative expression of K

关 键 词：KCNQ1重叠转录物1 微小RNA-199a-5p 人晶状体上皮细胞 凋亡 氧化应激 

分 类 号：R776.1[医药卫生—眼科]