[1]张新霞,陆丽红,狄文玉,等.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[J].眼科新进展,2020,40(8):727-730.[doi:10.13389/j.cnki.rao.2020.0165]
　ZHANG Xinxia,LU Lihong,DI Wenyu,et al.miRNA-140-5p modulates high glucose-induced oxidative stress of retinal pigment epithelial cells by targeting Nrf2[J].Recent Advances in Ophthalmology,2020,40(8):727-730.[doi:10.13389/j.cnki.rao.2020.0165]
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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/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
40卷
期数:
2020年8期
页码:
727-730
栏目:
实验研究
出版日期:
2020-08-05

文章信息/Info

Title:
miRNA-140-5p modulates high glucose-induced oxidative stress of retinal pigment epithelial cells by targeting Nrf2
作者:
张新霞; 陆丽红; 狄文玉; 王小敏; 王保君
453100　河南省新乡市,新乡医学院第一附属医院眼科（张新霞，陆丽红，王小敏，王保君）；453100　河南省新乡市,新乡医学院第一附属医院病理科（狄文玉）
Author(s):
ZHANG Xinxia1;  LU Lihong1;  DI Wenyu2;  WANG Xiaomin1;  WANG Baojun1
1.Department of Ophthalmology，the First Affiliated Hospital of Xinxiang Medical University,Xinxiang 453100, Henan Province, China
2.Department of Pathology，the First Affiliated Hospital of Xinxiang Medical University,Xinxiang 453100, Henan Province, China
关键词:
miR-140-5p; 核因子-类胡萝卜素2相关因子2; 高糖; 视网膜色素上皮细胞; 氧化应激
Keywords:
miR-140-5p;  nuclear factor erythroid 2-related factor 2;  high glucose;  retinal pigment epithelial cells;  oxidative stress
分类号:
R774
DOI:
10.13389/j.cnki.rao.2020.0165
文献标志码:
A
摘要:
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。
Abstract:
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.

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备注/Memo

备注/Memo:
N/A
更新日期/Last Update: 2020-08-05