中华皮肤科杂志 ›› 2021, Vol. 54 ›› Issue (7): 612-619.doi: 10.35541/cjd.20200871

• 论著 • 上一篇    下一篇

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

毛丽艳1    谢一航1    施辛1    张婷2    钱华2    吴亚芬2    鲁慧2    胡翠2    李巍2   

  1. 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
    第一作者现在苏州市吴中人民医院皮肤科  215128

  • 收稿日期:2020-09-01 修回日期:2021-04-16 发布日期:2021-07-02
  • 通讯作者: 施辛; 李巍 E-mail:shx9@163.com; carfield1981@126.com
  • 基金资助:
    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）

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

Mao Liyan1, Xie Yihang1, Shi Xin1, Zhang Ting2, Qian Hua2, Wu Yafen2, Lu Hui2, Hu Cui2, Li Wei2   

  1. 1Department of Dermatology, The Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu, China; 2Department of Dermatology, Children′s Hospital of Soochow University, Suzhou 215025, Jiangsu, China
    Mao Liyan is working at the Department of Dermatology, Suzhou Wuzhong People′s Hospital, Suzhou 215128, Jiangsu, China
  • Received:2020-09-01 Revised:2021-04-16 Published:2021-07-02
  • Contact: Shi Xin; Li Wei E-mail:shx9@163.com; carfield1981@126.com
  • Supported by:
    National Natural Science Foundation of China （81301380）; Basic Research Project of Suzhou Medical and Health Application （SYS2018076）

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摘要： 【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UVA组（t = 3.547，P < 0.05）。全基因组甲基化水平检测显示，上述各组甲基化水平（A450值）分别为0.676 ± 0.024、0.323 ± 0.043、0.506 ± 0.035、0.169 ± 0.024、0.602 ± 0.036、0.422 ± 0.029，各组间差异有统计学意义（F = 97.402，P < 0.01），其中UVA组低于空白对照组（P < 0.01），UVA + miR-26a-mimic低于miR-26a-mimic组（P < 0.01）和UVA组（P < 0.01），UVA + miR-26a-inhibitor组低于miR-26a-inhibitor组（P < 0.01），但高于UVA组（P < 0.05）。RT-PCR和Western 印迹显示，6组细胞间EZH2、DNMT1 mRNA和蛋白表达水平差异均有统计学意义（均P < 0.05），其中UVA组均低于空白对照组（P < 0.05），UVA + miR-26a mimic组均低于miR-26a mimic组（P < 0.05）和UVA 组（P < 0.05），而UVA + miR-26a inhibitor组均低于miR-26a inhibitor组（P < 0.05），但均高于UVA 组（P < 0.05）。结论 在UVA照射诱导皮肤成纤维细胞光老化中，miR-26a表达上调，细胞增殖活性下降，全基因组甲基化水平降低；上调miR-26a的表达，可下调其靶基因EZH2及甲基化相关基因DMNT1的表达，促进细胞光老化，反之，下调miR-26a的表达，可上调EZH2及DNMT1的表达，抑制细胞光老化。

关键词: 成纤维细胞, 紫外线, 细胞衰老, 微RNAs, 基因, EZH2, 微小RNA-26a

Abstract: 【Abstract】 Objective 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 difference in the expression of miR-26a between the two groups after 7 days of UVA irradiation （t = 5.295, P < 0.05）. Strong fluorescence signals were observed in the miR-26a mimic- or miR-26a inhibitor-transfected fibroblasts, suggesting a high transfection efficiency. Flow cytometry showed that the proportion of cells at G1 phase significantly differed among the blank control group, UVA group, miR-26a mimic group, UVA + miR-26a mimic group, miR-26a inhibitor group, and UVA + miR-26a inhibitor group （52.82% ± 2.56%, 78.56% ± 4.34%, 53.63% ± 3.13%, 89.52% ± 4.17%, 54.39% ± 3.86%, 65.34% ± 4.78%, respectively; F = 46.728, P < 0.01）, and significantly higher in the UVA group than in the blank control group （t = 8.848, P < 0.01）, higher in the UVA + miR-26a mimic group than in the miR-26a mimic group and UVA group （t = 11.922, 3.154, P < 0.01, < 0.05, respectively）, and higher in the UVA + miR-26a inhibitor group than in the miR-26a-inhibitor group （t = 3.087, P < 0.05）, but significantly lower in the UVA + miR-26a inhibitor group than in the UVA group （t = 3.547, P < 0.05）. Detection of the genome-wide methylation level showed that the methylation level （A450 value） significantly differed among the above groups （0.676 ± 0.024, 0.323 ± 0.043, 0.506 ± 0.035, 0.169 ± 0.024, 0.602 ± 0.036, 0.422 ± 0.029, respectively, F = 97.402, P < 0.01）, and significantly lower in the UVA group than in the blank control group （P < 0.01）, lower in the UVA + miR-26a mimic group than in the miR-26a mimic group and UVA group （both P < 0.01）, and lower in the UVA + miR-26a inhibitor group than in the miR-26a inhibitor group （P < 0.01）, but significantly higher in the UVA + miR-26a inhibitor group than in the UVA group （P < 0.05）. RT-PCR and Western blot analysis showed significant differences in the mRNA and protein expression of EZH2 and DNMT1 respectively among the 6 groups （both P < 0.05）, which were significantly lower in the UVA group than in the blank control group （P < 0.05）, lower in the UVA + miR-26a mimic group than in the miR-26a mimic group and UVA group （both P < 0.05）, and lower in the UVA + miR-26a inhibitor group than in the miR-26a inhibitor group （P < 0.05）, but significantly higher in the UVA + miR-26a inhibitor group than in the UVA group （P < 0.05）. Conclusion In the UVA irradiation-induced photoaging of skin fibroblasts, miR-26a expression was up-regulated, cellular proliferative activity and genome-wide methylation level decreased; up-regulation of miR-26a expression could down-regulate the expression of its target gene EZH2 and methylation-related gene DNM1, and promote cell photoaging, while down-regulation of miR-26a expression could up-regulate the expression of EZH2 and DNMT1, and inhibit cell photoaging.

Key words: Fibroblasts, Ultraviolet rays, Cell aging, MicroRNAs, Genes, EZH2, miR-26a