miR-223-3p对H_(2)O_(2)诱导的人表皮黑素细胞氧化应激反应和生物学功能的影响

Effect of micro-RNA-223 Expression on Oxidative Stress and Biological Function in Human Epidermal Melanocytes Induced by H_(2)O_(2)

目的 探讨miR-223-3p的表达对H_(2)O_(2)诱导的人表皮黑素氧化应激反应及生物学功能的影响。方法 建立H_(2)O_(2)诱导的人表皮黑素细胞氧化应激白癜风细胞模型,将miR-223-3p模拟物、miR-223-3p抑制剂以及阴性的对照物分别转染至人表皮黑素细胞氧化应激白癜风模型,并利用qRT-PCR测定miR-223-3p的表达;通过WST-1法和TBA法测定超氧化物歧化酶(SOD)活性和丙二醛(MDA)的含量活性;通过CCK-8法测定细胞增殖;流式细胞术测定细胞周期、凋亡和线粒体膜电位;使用生物信息软件预测FBXW7为miR-223-3p的潜在靶基因,并采用双荧光素酶报告基因实验加以证明。结果 成功确定了构建白癜风氧化应激细胞模型的H_(2)O_(2)的浓度为0.4 mmol/L,miR-223-3p模拟物可明显提高H_(2)O_(2)诱导的人表皮黑素细胞增殖率及SOD活性,改善细胞线粒体膜电位,降低细胞凋亡率及MDA和ROS含量,而miR-223-3p抑制剂组作用效应则相反。同时过表达miR-223-3p可降低氧化应激损伤的人表皮黑素细胞在G0/G1期的比例,提高在S期和G2/M期细胞的比例。同时验证了FBXW7是miR-223-3p的潜在靶基因。结论 过表达miR-223-3p可对人表皮黑素细胞H_(2)O_(2)诱导的氧化应激损伤起到一定的保护作用,在H_(2)O_(2)诱导的人表皮黑素细胞增殖和凋亡中发挥重要作用。

Objective To investigate the effect of the expression of miR-223-3p on oxidative and biological function of human epidermal melanin induced by H_(2)O_(2).Methods The cellular model of oxidative stress damage induced by H_2O_(2) was established.miR-223-3p mimic,miR-223-3p inhibitor and negative control were transfected into the model,and the expression of miR-223-3p was determined by qRT-PCR,the activities of SOD and MDA were determined by WST-1 method and TBA method.Cell proliferation was measured by CCK-8 method.Cell cycle,apoptosis and mitochondrial membrane potential were measured by flow cytometry.The bioinformatics software was used to predict that FBXW7 was a potential target gene of miR-223-3p,and it was verified by the experiment of double luciferase reporter gene.Results The oxidative stress cell model of vitiligo was successfully established,and the concentration of H_2O_(2) was 0.4 mmol/L.miR-223-3p mimic significantly increased the proliferation rate and SOD activity,improved the mitochondrial membrane potential,and decreased the apoptosis rate and MDA and ROS contents,while miR-223-3p inhibitor group had the opposite effect.At the same time,overexpression of miR-223-3p can reduce the proportion of human epidermal melanocytes injured by oxidative stress in G0/G1 phase,and increase the proportion of cells in S phase and G2/M phase.At the same time,it is verified that FBXW7 is a potential target gene of miR-223-3p.Conclusion Overexpression of miR-223-3p can protect human epidermal melanocytes from oxidative stress damage induced by H_(2)O_(2),and maybe play an important role in the proliferation and apoptosis of human epidermal melanocytes induced by H_(2)O_(2).