长链非编码RNA(lncRNA)KCNQ1重叠转录物1靶向抑制miR-628-5p在高糖诱导的视网膜色素上皮细胞损伤中的作用

Effect of targeted inhibition of miR-628-5p by long non-coding RNA KCNQ1 overlapping transcript 1 on high glucose-induced retinal pigment epithelial cell injury

目的探究长链非编码RNA(lncRNA)KCNQ1重叠转录物1(KCNQ1OT1)靶向抑制miR-628-5p在高糖诱导的视网膜色素上皮细胞损伤中的作用。方法采用实时荧光定量PCR(RT-qPCR)检测糖尿病视网膜病变(DR)、2型糖尿病(T2DM)、葡萄膜炎患者血清和房水KCNQ1OT1表达水平。用不同浓度葡萄糖处理人视网膜色素上皮细胞(ARPE-19),并检测KCNQ1OT1表达情况,选择最佳作用浓度。将ARPE-19细胞分为对照组(CON组)、高糖组(HG组)、HG+抑制物对照(si-NC)组、HG+KCNQ1OT1抑制物(si-KCNQ1OT1)组、HG+模拟物对照(miR-NC)组、HG+miR-628-5p组、HG+si-KCNQ1OT1+anti-miR-NC组、HG+si-KCNQ1OT1+anti-miR-628-5p组。RT-qPCR检测各组细胞KCNQ1OT1、miR-628-5p表达水平,流式细胞术检测细胞凋亡,ELISA检测各组细胞丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性,Western blot检测各组细胞活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved Caspase-3)蛋白表达水平,双荧光素酶报告实验检测KCNQ1OT1、miR-628-5p靶向关系。结果血清和房水中KCNQ1OT1表达水平:DR患者>T2DM患者>葡萄膜炎患者(均为P<0.05)。ARPE-19细胞中KCNQ1OT1表达水平:0 mmol·L^(-1)葡萄糖组<5 mmol·L^(-1)葡萄糖组<15 mmol·L^(-1)葡萄糖组<30 mmol·L^(-1)葡萄糖组(均为P<0.05),故选择30 mmol·L^(-1)葡萄糖进行实验。与CON组比较,HG组细胞KCNQ1OT1表达水平、细胞凋亡率、cleaved Caspase-3蛋白表达、MDA含量均显著升高,SOD活性、miR-628-5p显著降低(均为P<0.05)。与HG+si-NC组比较,HG+si-KCNQ1OT1组细胞KCNQ1OT1表达水平、细胞凋亡率、cleaved Caspase-3蛋白表达、MDA含量均显著降低,SOD活性显著升高(均为P<0.05)。与HG+miR-NC组比较,HG+miR-628-5P组细胞miR-628-5p、SOD活性均显著升高,细胞凋亡率、cleaved Caspase-3蛋白表达、MDA含量均显著降低(均为P<0.05)。与HG+si-KCNQ1OT1+anti-miR-NC组比较,HG+si-KCNQ1OT1+anti-miR-628-5p组细胞miR-628-5p、SOD活性均显著降低,细胞凋亡率、cleaved Caspase-3蛋白表达、MDA含量均显著升高(均为P<0.05)。结论lncRNA KCNQ1OT1通过靶向抑制miR-628-5p可以减轻高糖诱导的ARPE-19细胞凋亡和氧化损伤。

Objective To explore the effect of targeted inhibition of miR-628-5p by long non-coding RNA(lncRNA)KCNQ1 overlapping transcript 1(KCNQ1OT1)on high glucose(HG)-induced retinal pigment epithelial cell injury.Methods The real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was performed to detect KCNQ1OT1 expression in serum and aqueous humor of patients with diabetic retinopathy(DR),type 2 diabetes mellitus(T2DM)and uveitis.Human retinal pigment epithelial cells(ARPE-19)were treated with different concentrations of glucose,and their KCNQ1OT1 expression was detected to determine the optimal glucose concentration.ARPE-19 cells were divided into the control(CON)group,HG group,HG+si-NC group,HG+si-KCNQ1OT1 group,HG+miR-NC group,HG+miR-628-5p group,HG+si-KCNQ1OT1+anti-miR-NC group,and HG+si-KCNQ1OT1+anti-miR-628-5p group.The expression levels of KCNQ1OT1 and miR-628-5p in cells were measured by RT-qPCR.The cell apoptosis was detected by flow cytometry.The malondialdehyde(MDA)level and superoxide dismutase(SOD)activity were detected by enzyme-linked immunosorbent assay.The expression level of cleaved Caspase-3 protein was measured by Western blot.The targeting relationship between KCNQ1OT1 and miR-628-5p was detected by dual-luciferase reporter assay.Results The expression levels of KCNQ1OT1 in serum and aqueous humor of DR patients were higher than those of T2DM patients and uveitis patients,and those of uveitis patients were the lowest(all P<0.05).Compared with the 0 mmol·L^(-1) glucose group,the expression levels of KCNQ1OT1 in ARPE-19 cells in the 5 mmol·L^(-1) glucose group,15 mmol·L^(-1) glucose group,and 30 mmol·L^(-1) glucose group significantly increased,and those in the 30 mmol·L^(-1) glucose group were the highest(all P<0.05);thus,30 mmol·L^(-1) glucose was selected for subsequent experiments.Compared with the CON group,the KCNQ1OT1 expression,apoptosis rate,cleaved Caspase-3 protein expression,and MDA level in the HG group significantly increased,while the SOD activity and miR-628-5p significantly decreased(all P<0.05).Compared with the HG+si-NC group,the KCNQ1OT1 expression,apoptosis rate,cleaved Caspase-3 protein expression,and MDA level in the HG+si-KCNQ1OT1 group significantly decreased,while the SOD activity significantly increased(all P<0.05).Compared with the HG+miR-NC group,miR-628-5p and SOD activity in the HG+miR-628-5p group significantly increased,while the apoptosis rate,cleaved Caspase-3 protein expression,and MDA level significantly decreased(all P<0.05).Compared with the HG+si-KCNQ1OT1+anti-miR-NC group,miR-628-5p and SOD activity in the HG+si-KCNQ1OT1+anti-miR-628-5p group significantly decreased,while the apoptosis rate,cleaved Caspase-3 protein expression,and MDA level significantly increased(all P<0.05).Conclusion lncRNA KCNQ1OT1 can reduce the HG-induced apoptosis and oxidative damage of ARPE-19 cells by targeted inhibition of miR-628-5p.