中华皮肤科杂志 ›› 2020, Vol. 53 ›› Issue (6): 439-444.doi: 10.35541/cjd.20191062

• 论著 • 上一篇    下一篇

miRNA-1246靶向MAPK14在长波紫外线诱导人成纤维细胞光老化中机制的研究

胡翠    李巍    张婷    鲁慧    吴亚芳    钱华   

  1. 苏州大学附属儿童医院皮肤科  215025
  • 收稿日期:2019-11-10 修回日期:2020-04-07 发布日期:2020-06-01
  • 通讯作者: 钱华；李巍 E-mail:qianhua1980@gmail.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（SYS201644、SYS2018076）

Mechanism of miRNA-1246 targeting MAPK14 in ultraviolet A-induced photoaging of human fibroblasts

Hu Cui, Li Wei, Zhang Ting, Lu Hui, Wu Yafang, Qian Hua   

  1. Department of Dermatology, Children′s Hospital of Soochow University, Suzhou 215025, China
  • Received:2019-11-10 Revised:2020-04-07 Published:2020-06-01
  • Contact: Qian Hua; Li Wei E-mail:qianhua1980@gmail.com; carfield1981@126.com
  • Supported by:
    National Natural Science Foundation of China （81301380）; Suzhou Medical and Health Applied Basic Research Project （SYS201644, SYS2018076）

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摘要： 【摘要】 目的 探讨长波紫外线（UVA）诱导人成纤维细胞（HSF）光老化中miRNA-1246的表达及上调miR-1246表达对靶基因MAPK14及细胞老化的影响。方法 HSF分离自苏州大学附属儿童医院收集的健康儿童包皮环切术后皮肤标本，每日以10 J/cm2 UVA照射HSF，在初次照射及照射3、7、14 d采用实时定量PCR检测miR-1246的表达。将HSF细胞分为空白对照组、UVA组、miR-1246组、UVA + miR-1246组，通过慢病毒转染上调miR-1246的表达和照射UVA 14 d后，收集各组细胞，采用MTT检测细胞增殖活性，β半乳糖苷酶染色检测细胞老化，RT-PCR和Western印迹检测MAPK14、基质金属蛋白酶1（MMP-1）的表达。多组间均数的比较用单因素方差分析，组间两两比较采用LSD-t检验。结果 在照射7 d、14 d时，UVA组HSF的miR-1246相对表达水平（4.69 ± 0.85、3.59 ± 0.45）均低于空白对照组（8.42 ± 0.75、7.61 ± 0.49），差异均有统计学意义（t = 29.84、31.93，均P < 0.01）。上调miR-1246的表达和照射UVA后，MTT结果显示，空白对照组、UVA组、miR-1246组、UVA + miR-1246组细胞增殖活性（0.82 ± 0.03、0.23 ± 0.02、0.81 ± 0.02、0.61 ± 0.02）差异有统计学意义（F = 34.90，P < 0.05），UVA组低于空白对照组（t = 28.14，P < 0.01），UVA + miR-1246组低于miR-1246组（t = 10.61，P < 0.01），高于UVA组（t = 20.30，P < 0.01）。β半乳糖苷酶染色检测显示，4组老化细胞阳性率（3.93% ± 1.11%、81.29% ± 2.53%、5.50% ± 1.15%、54.13% ± 2.09%）差异有统计学意义（F = 16.14，P < 0.05），其中UVA组高于空白对照组（t = 48.46，P < 0.01），而UVA + miR-1246组高于miR-1246组（t = 35.31，P < 0.01），低于UVA组（t = 14.32，P < 0.01）。RT-PCR和Western 印迹均显示，4组细胞MAPK14、MMP-1的mRNA、蛋白相对水平差异均有统计学意义（均P < 0.05），其中UVA组高于空白对照组（P < 0.05），UVA + miR-1246组高于miR-1246组（P < 0.05），低于UVA组（P < 0.05）。结论 UVA照射诱导的HSF光老化中，miR-1246的表达受到抑制，上调miR-1246的表达可抑制其靶基因MAPK14及老化相关蛋白MMP-1的表达，起到抗细胞光老化的作用。

关键词: 成纤维细胞, 紫外线, 微RNAs, 丝裂原活化蛋白激酶14, 基质金属蛋白酶1, miR-1246

Abstract: 【Abstract】 Objective To investigate the miRNA-1246 expression in photoaged human fibroblasts （HSFs） induced by ultraviolet A （UVA）, and to evaluate the effect of upregulating miRNA-1246 expression on its target gene MAPK14 and cell aging. Methods HSFs were isolated from foreskins of healthy children after circumcision in Children′s Hospital of Soochow University, and irradiated with 10 J/cm2 UVA once a day for 14 consecutive days. Real-time quantitative PCR was performed to determine the expression of miR-1246 immediately after the first irradiation and on days 3, 7 and 14 after the start of irradiation. Some HSFs were divided into 4 groups: blank control group receiving no treatment, UVA group irradiated with 10 J/cm2 UVA for 14 days, miR-1246 group transfected with a lentiviral vector carrying miR-1246, and UVA + miR-1246 group transfected with a lentiviral vector carrying miR-1246 followed by irradiation with UVA. After treatment, the HSFs were collected, methyl thiazolyl tetrazolium （MTT） assay was performed to assess cellular proliferativy activity, β-galactosidase staining to detect senescent cells, RT-PCR and Western blot analysis were conducted to measure the mRNA and protein expression of MAPK14 and matrix metalloproteinase 1 （MMP-1）. One-way analysis of variance was used for comparison of means among multiple groups, and least significant difference （LSD）-t test was used for multiple comparisons. Results On days 7 and 14, the relative expression of miR-1246 in HSFs was significantly lower in the UVA group （4.69 ± 0.85, 3.59 ± 0.45, respectively） than in the blank control group （8.42 ± 0.75, 7.61 ± 0.49, t = 29.84, 31.93, respectively, both P < 0.01）. After upregulation of miR-1246 and irradiation with UVA, MTT assay showed that the cellular proliferative activity significantly differed among the blank control group, UVA group, miR-1246 group, UVA + miR-1246 group （0.82 ± 0.03, 0.23 ± 0.02, 0.81 ± 0.02, 0.61 ± 0.02, respectively; F = 34.90, P < 0.05）, significantly lower in the UVA group than in the blank control group （t = 28.14, P < 0.01）, lower in the UVA + miR-1246 group than in the miR-1246 group （t = 10.61, P < 0.01）, but significantly higher in the UVA + miR-1246 group than in the UVA group （t = 20.30, P < 0.01）. β-Galactosidase staining showed that the proportion of senescent cells significantly differed among the above 4 groups （3.93% ± 1.11%, 81.29% ± 2.53%, 5.50% ± 1.15%, 54.13% ± 2.09%, respectively; F = 16.14, P < 0.05）, significantly higher in the UVA group than in the blank control group （t = 48.46, P < 0.01）, higher in the UVA + miR-1246 group than in the miR-1246 group （t = 35.31, P < 0.01）, but significantly lower in the UVA + miR-1246 group than in the UVA group （t = 14.32, P < 0.01）. Both RT-PCR and Western blot analysis showed that the mRNA and protein expression of MAPK14 and MMP-1 significantly differed among the above 4 groups （both P < 0.05）, significantly higher in the UVA group than in the blank control group （P < 0.05）, higher in the UVA + miR-1246 group than in the miR-1246 group （P < 0.05）, but significantly lower in the UVA + miR-1246 group than in the UVA group （P < 0.05）. Conclusions In the senescent HSFs induced by UVA, the expression of miR-1246 is suppressed. Upregulating the expression of miR-1246 can exert anti-photoaging effect by inhibiting the expression of its target gene MAPK14 and aging-related protein MMP-1.

Key words: Fibroblasts, Ultraviolet rays, MicroRNAs, Mitogen-activated protein kinase 14, Matrix metalloproteinase 1, miR-1246

引用本文

胡翠 李巍 张婷 鲁慧 吴亚芳 钱华. miRNA-1246靶向MAPK14在长波紫外线诱导人成纤维细胞光老化中机制的研究[J]. 中华皮肤科杂志, 2020,53(6):439-444. doi:10.35541/cjd.20191062

Hu Cui, Li Wei, Zhang Ting, Lu Hui, Wu Yafang, Qian Hua. Mechanism of miRNA-1246 targeting MAPK14 in ultraviolet A-induced photoaging of human fibroblasts[J]. Chinese Journal of Dermatology, 2020, 53(6): 439-444.doi:10.35541/cjd.20191062