摘要
目的探讨miR-181a对过氧化氢(H_(2)O_(2))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(0μmol·L^(-1)H_(2)O_(2))、200μmol·L^(-1)H_(2)O_(2)组、400μmol·L^(-1)H_(2)O_(2)组、600μmol·L^(-1)H_(2)O_(2)组,分别使用相应浓度H_(2)O_(2)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,200μmol·L^(-1)H_(2)O_(2)组、400μmol·L^(-1)H_(2)O_(2)组、600μmol·L^(-1)H_(2)O_(2)组细胞存活率均降低(均为P<0.05)。细胞miR-181a mRNA的表达随着H_(2)O_(2)浓度的增加而增加,SIRT1蛋白的表达随着H_(2)O_(2)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H_(2)O_(2)诱导的HTMCs氧化应激作用。
Objective To investigate the regulatory effect of miR-181a on H_(2)O_(2)-induced oxidative stress in trabecular meshwork cells and its possible mechanism.Methods Human trabecular meshwork cells(HTMCs)were induced with 0μmol·L^(-1),200μmol·L^(-1),400μmol·L^(-1)or 600μmol·L^(-1)H_(2)O_(2),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 200μmol·L^(-1),400μmol·L^(-1),and 600μmol·L^(-1)H_(2)O_(2) significantly decreased survival rate in HTMCs(all P<0.05).Besides,miR-181a was upregulated,while SIRT1 was downregulated with the increase in H_(2)O_(2) 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 H_(2)O_(2)-induced oxidative stress of HTMCs by targeting SIRT1.
作者
田思佳
王骞
张蕾
屈林
肖燕
朱俊英
王怀洲
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)
出处
《眼科新进展》
CAS
北大核心
2021年第9期826-830,共5页
Recent Advances in Ophthalmology
基金
河南省医学科技攻关计划项目(编号LHGJ201901012)。
