[1]田思佳,王骞,张蕾,等.miR-181a靶向沉默信息调节因子相关酶1在过氧化氢诱导的人小梁网细胞氧化应激中的调节作用[J].眼科新进展,2021,41(9):826-830.[doi:10.13389/j.cnki.rao.2021.0173]
　TIAN Sijia,WANG Qian,ZHANG Lei,et al.miR-181a regulates H2O2-induced oxidative stress in human trabecular meshwork cells by targeting SIRT1[J].Recent Advances in Ophthalmology,2021,41(9):826-830.[doi:10.13389/j.cnki.rao.2021.0173]
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miR-181a靶向沉默信息调节因子相关酶1在过氧化氢诱导的人小梁网细胞氧化应激中的调节作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
41卷
期数:
2021年9期
页码:
826-830
栏目:
实验研究
出版日期:
2021-09-05

文章信息/Info

Title:
miR-181a regulates H2O2-induced oxidative stress in human trabecular meshwork cells by targeting SIRT1
作者:
田思佳; 王骞; 张蕾; 屈林; 肖燕; 朱俊英; 王怀洲
450000　河南省郑州市，郑州市第二人民医院眼科
Author(s):
TIAN Sijia; WANG Qian; ZHANG Lei; QU Lin; XIAO Yan; ZHU Junying; WANG Huaizhou
Department of Ophthalmology,Zhengzhou Second People’s Hospital,Zhengzhou 450000,Henan Province,China
关键词:
青光眼; 人小梁网细胞; 氧化应激; miR-181a; 沉默信息调节因子相关酶1
Keywords:
glaucoma;  human trabecular meshwork cells;  oxidative stress;  miR-181a;  silent information regulator factor related enzymes 1
分类号:
R775
DOI:
10.13389/j.cnki.rao.2021.0173
文献标志码:
A
摘要:
目的　探讨miR-181a对过氧化氢（H2O2）诱导的人小梁网细胞（HTMCs)氧化应激的调节作用及其机制。方法　HTMCs随机分为空白组（0 μmol·L-1 H2O2）、200 μmol·L-1 H2O2组、400 μmol·L-1 H2O2组、600 μmol·L-1 H2O2组，分别使用相应浓度H2O2处理HTMCs，24 h后MTT法检测各组细胞存活率，实时荧光定量PCR（qRT-PCR）检测各组细胞miR-181a mRNA表达，Western blot检测各组细胞中沉默信息调节因子相关酶1（SIRT1）蛋白表达。根据转染物的不同分为miR-181a inhibitor组、miR-181a NC组、miR-181a mimics组、miR-181a inhibitor+si-SIRT1组和miR-181a inhibitor+si-NC组，使用二氯二氢荧光素-乙酰乙酸酯（DCFH-DA）荧光探针检测各组细胞内活性氧（ROS）含量，使用超氧化物歧化酶（SOD）和丙二醛（MDA）ELISA试剂盒检测各组细胞SOD和MDA活性，使用Annexin V FITC/PI联合流式细胞仪检测细胞凋亡情况，Western blot检测各组细胞中SIRT1蛋白表达，双荧光素酶报告基因实验对靶点进行验证。结果　与空白组比较，200 μmol·L-1 H2O2组、400 μmol·L-1 H2O2组、600 μmol·L-1 H2O2组细胞存活率均降低（均为P<0.05）。细胞miR-181a mRNA的表达随着H2O2浓度的增加而增加，SIRT1蛋白的表达随着H2O2浓度的增加而降低（均为P<0.05）。与miR-181a NC组比较，miR-181a mimics组细胞凋亡率、MDA活性和ROS含量均升高，细胞SOD活性、SIRT1蛋白表达均降低，miR-181a inhibitor组细胞凋亡率、MDA活性和ROS含量均降低，细胞SOD活性、SIRT1蛋白表达均升高（均为P<0.05）。双荧光素酶报告基因实验证实，miR-181a靶向抑制SIRT1蛋白的表达。与miR-181a inhibitor+si-NC组比较，miR-181a inhibitor+si-SIRT1组细胞凋亡率、MDA活性和ROS含量均升高，SOD活性降低（均为P<0.05）。结论　miR-181a可能通过靶向SIRT1调节H2O2诱导的HTMCs氧化应激作用。
Abstract:
Objective　To investigate the regulatory effect of miR-181a on H2O2-induced oxidative stress in trabecular meshwork cells and its possible mechanism.Methods　Human trabecular meshwork cells (HTMCs) were induced with 0 μmol·L-1, 200 μmol·L-1, 400 μmol·L-1 or 600 μmol·L-1 H2O2, respectively,for 24 h. MTT assay was performed to detect cell viability. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA level of miR-181a, and Western blot was used to detect protein level of silent information regulator factor related enzymes 1 (SIRT1). In addition, HTMCs were transfected with miR-181a inhibitor, miR-181a negative control, miR-181a mimics, SIRT1 small interfering RNA (si-SIRT1) or negative control (si-NC) was transfected into HTMCs cells. DCFH-DA fluorescent probe was used to detect the intracellular reactive oxygen species (ROS) levels in each transfection group. Superoxide dismutase (SOD) and malondialdehyde (MDA) activities were measured by ELISA. Cell apoptosis was determined by Annexin V FITC/PI using flow cytometry. Western blot was used to detect protein level of SIRT1 in each transfection group. Dual-luciferase reporter assay was performed to identify the targeting between miR-181a and SIRT1.Results　Compared with the blank group, induction of 200 μmol·L-1, 400 μmol·L-1, and 600 μmol·L-1 H2O2 significantly decreased survival rate in HTMCs (all P<0.05). Besides, miR-181a was upregulated, while SIRT1 was downregulated with the increase in H2O2 concentration (all P<0.05). Compared with the miR-181a NC group, transfection of miR-181a mimics increased the apoptosis rate, and MDA and ROS activities, but decreased SOD activity and downregulated SIRT1. Knockdown of miR-181a yielded the opposite results (all P<0.05). Dual-luciferase reporter assay confirmed that miR-181a targeted SIRT1. Compared with HTMCs with miR-181a knockdown, those with co-silence of miR-181 and SIRT1 presented higher apoptosis rate, and MDA and ROS activities, but lower SOD activity (all P<0.05).Conclusion　MiR-181a may regulate H2O2-induced oxidative stress of HTMCs by targeting SIRT1.

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备注/Memo

备注/Memo:
河南省医学科技攻关计划项目（编号：LHGJ201901012）
更新日期/Last Update: 2021-09-05