中华皮肤科杂志 ›› 2015, Vol. 48 ›› Issue (6): 416-420.

• 论著 • 上一篇    下一篇

miR-145对人角质形成细胞系HaCaT细胞增殖、凋亡和细胞周期的影响

李璟蓉1,王建琴2,方锐华3,曾仁山3,莫金雪1,郭云龙1,贾淑青1   

  1. 1. 广州市南沙中心医院
    2. 广州市第一人民医院广州市南沙中心医院
    3. 广州市第一人民医院皮肤科
  • 收稿日期:2014-10-13 修回日期:2015-03-16 出版日期:2015-06-15 发布日期:2015-06-03
  • 通讯作者: 王建琴 E-mail:jianqinwang@tom.com
  • 基金资助:

    广东省医学科研基金

Effects of miR-145 on the proliferation, apoptosis and cell cycle of a human keratinocyte cell line HaCaT

  • Received:2014-10-13 Revised:2015-03-16 Online:2015-06-15 Published:2015-06-03
  • Contact: Jian-Qin WANG E-mail:jianqinwang@tom.com

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摘要：

目的 研究miR-145对人角质形成细胞系HaCaT细胞增殖、细胞周期及凋亡的调控效应。 方法化学合成miR-145的模拟物，采用瞬时转染的方法过表达miR-145。采用实时PCR方法检测miR-145的表达。MTS方法检测过表达miR-145对HaCaT细胞增殖的影响。流式细胞仪分析过表达miR-145对细胞周期及凋亡的影响。采用荧光素酶实验，实时PCR和Western印迹鉴定NRAS是否为miR-145的靶基因。 结果 与阴性对照（NC）mimics转染组相比，miR-145 mimics转染组miR-145表达水平上调（85.00 ± 1.21）倍，两组差异有统计学意义（t = 115.90，P < 0.001）。转染miR-145 mimics有抑制HaCaT细胞的增殖作用（F = 8.76，P = 0.008）；转染后的时间因素（24、48、72、96 h）对细胞有影响（F = 17.85，P < 0.001），转染mimics和培养时间之间不存在交互作用（F = 1.21，P = 0.18）。与NC mimics转染组相比，miR-145 mimics转染组的早期凋亡、晚期凋亡细胞比例均明显增加，差异有统计学意义（18.9% ± 4.1%比4.3% ± 1.2%，t = 7.126，P < 0.01；9.3% ± 2.3%比3.6% ± 1.6%，t = 12.38，P < 0.01）。与NC mimics转染组相比，miR-145 mimics转染组HaCaT细胞的G2及S期细胞比例均明显降低，差异均有统计学意义（6.26% ± 1.2%比19.36% ± 3.45%，t =7.610，P = 0.017；7.91% ± 1.3%比18.56% ± 5.23%，t = 7.230，P = 0.019），而处于G1期的细胞比例升高，差异也有统计学意义（85.83% ± 5.2%比62.08% ± 6.23%，t = 11.78，P = 0.007）。与NC mimics联合psi-CHECK2-NRAS-wild组相比，在293T细胞中共转染miR-145 mimics和psi-CHECK2-NRAS-wild质粒，其荧光素酶值明显下降，miR-145可抑制含NRAS mRNA 3′UTR报告基因的荧光素酶表达（t = 11.09，P = 0.008）；而将NRAS mRNA 3′UTR报告基因上miR-145的结合位点进行突变后，转染miR-145 mimics对含NRAS mRNA 3′UTR报告基因的荧光素酶表达无明显的影响（P > 0.05）。实时PCR和Western印迹结果表明，过表达miR-145 mimics后，NRAS mRNA表达水平未出现明显变化（P > 0.05），对NRAS蛋白水平的表达有明显的抑制（1.52 ± 0.07比0.20 ± 0.02，t = 28.43，P < 0.01）。 结论 miR-145可能通过NRAS影响HaCaT细胞的周期从而抑制细胞的增殖，同时促进HaCaT细胞的凋亡。

Abstract:

Li Jingrong*, Wang Jianqin, Fang Ruihua, Zeng Renshan, Mo Jinxue, Guo Yunlong, Jia Shuqing. *Department of Dermatology, Nansha Central Hospital of Guangzhou, Guangzhou 511457, China Corresponding author: Wang Jianqin, Email: jianqinwang @tom.com 【Abstract】 Objective To investigate the regulatory effects of miR-145 on the proliferation, cell cycle and apoptosis of a human keratinocyte cell line HaCaT. Methods miR-145 mimics and negative control （NC） mimics were chemically synthesized and then transiently transfected into HaCaT cells respectively. After additional culture for different durations, real-time PCR was performed to determine the expression level of miR-145, MTS assay to estimate cell proliferation, and flow cytometry to detect cell apoptosis and cycle. Luciferase assay, real-time PCR and Western blot were conducted to determine whether NRAS was the target gene of miR-145. Results The miR-145 expression level in miR-145 mimic-transfected cells increased by 85.00 ± 1.21 folds compared with NC mimic-transfected cells （t = 115.90, P < 0.0001）. The transfection with miR-145 mimics significantly inhibited the proliferation of HaCaT cells （F = 8.76, P = 0.008）, and the inhibitory effect significantly varied with the duration （24 － 96 hours） of culture after transfection, with no interaction effect between the transfection with miR-145 mimics and culture duation （F = 1.21, P = 0.18）. Compared with NC mimic-transfected cells, those transfected with miR-145 mimics showed a significant increase in the proportion of early apoptotic cells （18.9% ± 4.1% vs. 4.3% ± 1.2%, t = 7.126, P < 0.01）, late apoptotic cells （9.3% ± 2.3% vs. 3.6% ± 1.6%, t = 12.38, P < 0.01）, G1-phase cells （85.83% ± 5.2% vs. 62.08% ± 6.23%, t = 11.78, P = 0.007）, but a significant decrease in the percentage of G2-phase cells （6.26% ± 1.2% vs. 19.36% ± 3.45%, t =7.610, P = 0.017） and S-phase cells （7.91% ± 1.3% vs. 18.56% ± 5.23%, t = 7.230, P = 0.019）. As luciferase assay showed, luciferase activity was significantly lower in HaCaT cells cotransfected with miR-145 mimics and a recombinant luciferase reporter vector psi-CHECK2-NRAS-wild carrying the wild-type 3′UTR of NRAS than in those cotransfected with NC mimics and the vector psi-CHECK2-NRAS-wild （t = 11.09, P = 0.008）, but similar between cells cotransfected with miR-145 mimics and a recombinant luciferase reporter vector psi-CHECK2-NRAS-mut carrying the mutant-type 3′UTR of NRAS and those cotransfected with NC mimics and the vector psi-CHECK2-NRAS-mut （P > 0.05）. Real-time PCR and Western blot revealed that the overexpression of miR-145 mimics had no significant effect on NRAS mRNA expression （P > 0.05）, but significantly inhibited NRAS protein expression （1.52 ± 0.07 vs. 0.20 ± 0.02, t = 28.43, P < 0.01）. Conclusion miR-145 might inhibit proliferation and promote apoptosis of HaCaT cells by influencing cell cycle via NRAS.