miR-181 a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

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(0μmol·L-1 H2 O2)、200μmol·L-1 H2 O2组、400μmol·L-1 H2 O2组、600μmol·L-1 H2 O2组,分别使用相应浓度H2 O2处理H...

miR-181a regulates H2O2-induced oxidative stress in human trabecular meshwork cells by targeting SIRT1

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.