miR-26a靶向EZH2在长波紫外线诱导人皮肤成纤维细胞光老化中的作用机制研究  

Role and action mechanism of microRNA-26a targeting EZH2 in ultraviolet A-induced photoaging of human skin fibroblasts

作　　者：毛丽艳 谢一航 施辛[1] 张婷[2] 钱华[2] 吴亚芬[2] 鲁慧[2] 胡翠[2] 李巍[2] Mao Liyan;Xie Yihang;Shi Xin;Zhang Ting;Qian Hua;Wu Yafen;Lu Hui;Hu Cui;Li Wei(Department of Dermatology,The Second Affiliated Hospital of Soochow University,Suzhou 215004,Jiangsu,China;Department of Dermatology,Children′s Hospital of Soochow University,Suzhou 215025,Jiangsu,China)

机构地区：[1]苏州大学附属第二医院皮肤科,215004 [2]苏州大学附属儿童医院皮肤科,215025

出　　处：《中华皮肤科杂志》2021年第7期612-619,共8页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81301380);苏州市医疗卫生应用基础研究项目(SYS2018076)。

摘　　要：目的探讨长波紫外线诱导人皮肤成纤维细胞光老化中miRNA-26a的表达情况,以及上调或下调miR-26a表达对全基因组甲基化水平、靶基因组蛋白-赖氨酸N-甲基转移酶2(EZH2)及细胞老化的影响。方法以10 J/cm2 UVA照射人皮肤成纤维细胞,分别在第0、3、7天提取RNA,实时定量反转录PCR(RT-PCR)检测miR-26a的表达;通过转染miR-26a模拟物(mimic)和miR-26a抑制物(inhibitor)上调或下调miR-26a的表达,通过荧光显微镜观察和实时定量PCR检测miR-26a表达量,并评估转染效率。将人皮肤成纤维细胞分为空白对照组(不做任何处理)、UVA组(UVA照射)、miR-26a-mimic组(转染miR-26a-mimic)、UVA+miR-26a-mimic组(转染miR-26a-mimic后UVA照射)、miR-26a-inhibitor组(转染miR-26a-inhibitor)、UVA+miR-26a-inhibitor组(转染miR-26a-inhibitor后UVA照射)。第7天完成UVA照射后,收集各组细胞,采用流式细胞仪检测细胞周期,DNA甲基化定量检测试剂盒检测全基因组甲基化水平,RT-PCR检测EZH2、DNA甲基转移酶1(DNMT1)mRNA以及miR-26a的表达,Western印迹检测EZH2以及DNMT1蛋白的表达。采用单因素方差分析及LSD-t检验进行统计学分析。结果随着培养时间的延长,对比空白对照组,UVA照射组中miR-26a表达量逐渐增高,在UVA照射第7天后,两组间miR-26a表达水平差异有统计学意义(t=5.295,P<0.05)。miR-26a mimic/miR-26a inhibitor转染HSF后,细胞内均有较高的荧光表达,提示均具有较高的转染效率。流式细胞仪检测显示,空白对照组、UVA组、miR-26a-mimic组、UVA+miR-26a-mimic组、miR-26a-inhibitor组、UVA+miR-26a-inhibitor组G1期细胞比例分别为52.82%±2.56%、78.56%±4.34%、53.63%±3.13%、89.52%±4.17%、54.39%±3.86%、65.34%±4.78%,各组间差异有统计学意义(F=46.728,P<0.01),其中UVA组高于空白对照组(t=8.848,P<0.01),UVA+miR-26a-mimic高于miR-26a-mimic组(t=11.922,P<0.01)和UVA组(t=3.154,P<0.05),UVA+miR-26a-inhibitor组高于miR-26a-inhibitor组(t=3.087,P<0.05),但低于UObjective To investigate the expression of microRNA(miR)-26a in human skin fibroblasts during photoaging induced by ultraviolet A(UVA),and to evaluate the effect of up-or down-regulation of miR-26a expression on the methylation level of the whole genome,the target gene enhancer of zeste homolog 2(EZH2)and cell aging.Methods Some human skin fibroblasts were irradiated with 10 J/cm2 UVA once a day for 7 consecutive days,RNA was extracted on days 0,3 and 7,and real-time quantitative reverse PCR(RT-PCR)was performed to determine the expression of miR-26a;miR-26a mimics and inhibitors were transfected into fibroblasts to up-or down-regulate the expression of miR-26a respectively,and fluorescence microscopy and RT-PCR were performed to determine the expression of miR-26a and evaluate the transfection efficiency.Some human skin fibroblasts were divided into 6 groups:blank control group receiving no treatment,UVA group treated with UVA irradiation according to the above method,miR-26a mimic group transfected with miR-26a-mimics,UVA+miR-26a mimic group transfected with miR-26a-mimics followed by UVA irradiation,miR-26a inhibitor group transfected with miR-26a inhibitors,UVA+miR-26a inhibitor group transfected with miR-26a inhibitors followed by UVA irradiation.On day 7,cells in each group were collected after the end of UVA irradiation.Then,flow cytometry was performed to detect cell cycle,DNA methylation quantitative detection kit was used to detect the methylation level of whole genome,RT-PCR was conducted to determine the mRNA expression of EZH2(a histone-lysine N-methyltransferase enzyme),DNA methyltransferase 1(DNMT1)and miR-26a,and Western blot analysis was performed to determine the protein expression of EZH2 and DNMT1.Statistical analysis was carried out by using one-way analysis of variance and least significant difference-t test.Results Compared with the unirradiated control group,the expression of miR-26a gradually increased in the UVA irradiation group over time during the culture,and there was a significant d

关 键 词：成纤维细胞 紫外线 细胞衰老 微RNAS 基因 EZH2 微小RNA-26a 

分 类 号：R751[医药卫生—皮肤病学与性病学]