miR-140-5p靶向Nrf2对高糖诱导的视网膜色素上皮细胞氧化应激的调节作用

miRNA-140-5p modulates high glucose-induced oxidative stress of retinal pigment epithelial cells by targeting Nrf2

目的探讨miR-140-5p靶向核因子-类胡萝卜素2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)对高糖诱导的视网膜色素上皮(retinal pigment epithelial,RPE)细胞氧化应激的调节作用及对应的分子生物学机制。方法使用5 mmol·L^-1、10 mmol·L^-1、20 mmol·L^-1、30 mmol·L^-1、50 mmol·L^-1的葡萄糖分别处理ARPE-19细胞。利用CCK-8法及RT-qPCR检测各葡萄糖浓度下ARPE-19细胞的活力及miR-140-5p、Nrf2 mRNA表达情况。将miR-140-5p inhibitor、miR-140-5p-NC、miR-140-5p-mimic、si-Con或si-Nrf2转染入ARPE-19细胞,根据转染物和葡萄糖浓度不同分组,使用DCFH-DA荧光探针检测各组细胞内活性氧(reactive oxygen species,ROS)水平,使用超氧化物歧化酶(superoxide dismutase,SOD)活性试剂盒检测SOD活性;使用RT-qPCR检测不同处理的ARPE-19细胞Nrf2 mRNA表达情况,并通过荧光素酶报告基因实验对靶点进行验证。结果与葡萄糖浓度为5 mmol·L^-1时相比,30 mmol·L^-1、50 mmol·L^-1葡萄糖作用下ARPE-19细胞存活率显著降低(P<0.05、P<0.001),而miR-140-5p相对表达量则明显升高(P<0.05、P<0.001)。与50 mmol·L^-1葡萄糖+miR-140-5p-NC处理组相比,50 mmol·L^-1葡萄糖+miR-140-5p inhibitor处理组细胞存活率显著增加,ROS含量显著降低,SOD活性显著增加(均为P<0.01)。与葡萄糖浓度为5 mmol·L^-1时相比,30 mmol·L^-1、50 mmol·L^-1葡萄糖作用下ARPE-19细胞Nrf2 mRNA相对表达量均显著降低(P<0.01、P<0.001)。荧光素酶报告基因实验结果显示,与转染miR-140-5p-NC组相比,转染miR-140-5p-mimic组Nrf2相对荧光素酶活性显著降低,转染miR-140-5p-inhibitor组Nrf2相对荧光素酶活性显著增多,差异均有统计学意义(均为P<0.001)。在50 mmol·L^-1葡萄糖浓度下,与转染miR-140-5p-inhibitor+si-Con的ARPE-19细胞比较,转染miR-140-5p inhibitor+si-Nrf2后ARPE-19细胞存活率降低,细胞内ROS含量增高,SOD活性下降(均为P<0.01)。结论miR-140-5p可能通过靶向Nrf2调节高糖诱导的RPE细胞氧化应激作用。

Objective To investigate the molecular mechanism of oxidative stress induced by miR-140-5p in retinal pigment epithelial cells(RPE)induced by high glucose(HG)by targeting nuclear factor erythroid 2-related factor 2(Nrf2).Methods ARPE-19 cells were treated with glucose at concentrations of 5 mmol·L^-1,10 mmol·L^-1,20 mmol·L^-1,30 mmol·L^-1,50 mmol·L^-1 respectively.CCK-8 was used to detect the survival rate and RT-qPCR was applied to detect miR-140-5p mRNA expression of RPE cells with increasing glucose concentration in turn;DCFH-DA fluorescence probe was used to detect the level of reactive oxygen species(ROS)in cells treated with interfering miR-140-5p,MDA and SOD enzyme activity kit were used to detect superoxide dismutase(SOD)activities,Nrf2 mRNA and protein expression were detected by RT-PCR and Western blot,and the target was verified by luciferase reporter gene experiment.Results Compared with the low glucose condition of 5 mmol·L^-1,the survival rate of RPE cells of 30 mmol·L^-1 and 50 mmol·L^-1 decreased significantly(P<0.05,P<0.001)and the expression level of miR-140-5p increased significantly(P<0.05,P<0.001).Compared with the 50 mmol·L^-1 glucose control group,the transfection of miR-140-5p inhibitor significantly increased cell survival rate,decreased ROS content and increased SOD activity(all P<0.01).Compared with the low glucose conditions of 5 mmol·L^-1,the Nrf2 mRNA expression levels of RPE cells of 30 mmol·L^-1 and 50 mmol·L^-1 decreased significantly(P<0.01,P<0.001).The results of luciferase test showed that,compared with the empty control group,the transfection of miR-140-5p mimic significantly decreased the Nrf2 activity,whereas the transfection of miR-140-5p inhibitor significantly increased the Nrf2 activity,with significant difference(both P<0.001).At 50 mmol·L^-1 glucose concentration,compared with the RPE cells transfected with miR-140-5p-inhibitor+si-Con,the RPE cells transfected with miR-140-5p inhibitor and si-Nrf2 significantly decreased cell survival rate,decreased SOD activity,and increased ROS content(all P<0.01).Conclusion miR-140-5p can modulate high glucose-induced oxidative stress injury of RPE cells by targeting Nrf2.