中华皮肤科杂志 ›› 2021, Vol. 54 ›› Issue (6): 499-503.doi: 10.35541/cjd.20200707

• 论著 • 上一篇    下一篇

微RNA-125a靶向白细胞介素23受体信号通路抑制HaCaT细胞增殖机制的初步研究

苏芳1    晋亮2    李浩1    丁英洁1    孙晓杰1    孙晓冬1    刘玮2    徐桂娟1    王强1    刘永斌1   

  1. 1沈阳市第七人民医院皮肤科  110003；2空军特色医学中心皮肤科，北京  100142
  • 收稿日期:2020-07-13 修回日期:2020-12-14 发布日期:2021-05-31
  • 通讯作者: 刘玮；刘永斌 E-mail:lwei5811@126.com; syqyybin@126.com
  • 基金资助:
    辽宁省科学技术计划项目（2019?ZD?0975）；沈阳市中青年科技创新人才支持计划项目（RC200417）

Mechanisms underlying microRNA-125a-mediated inhibition of proliferation of HaCaT cells by targeting the interleukin 23 receptor signaling pathway: a preliminary study

Su Fang1, Jin Liang2, Li Hao1, Ding Yingjie1, Sun Xiaojie1, Sun Xiaodong1, Liu Wei2, Xu Guijuan1, Wang Qiang1, Liu Yongbin1   

  1. 1Department of Dermatology, The Seventh People′s Hospital of Shenyang, Shenyang 110003, China; 2Department of Dermatology, Air Force Medical Center, Beijing 100142, China
  • Received:2020-07-13 Revised:2020-12-14 Published:2021-05-31
  • Contact: Liu Wei; Liu Yongbin E-mail:lwei5811@126.com; syqyybin@126.com
  • Supported by:
    Science and Technology Planning Project of Liaoning Province（2019?ZD?0975）; Young and Middle?aged Scientific and Technological Innovation Talent Support Program of Shenyang（RC200417）

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摘要： 【摘要】 目的 探讨微RNA（miR）-125a抑制角质形成细胞增殖的相关机制。方法 用白细胞介素（IL）-23干预处理人永生化角质形成细胞（HaCaT）24 h后，分为miR-125a组和miR-NC组，分别转染miR-125a过表达质粒和过表达对照质粒。采用细胞计数试剂盒（CCK8）法检测两组转染后0、24、48、72 h HaCaT细胞增殖能力，采用实时荧光定量PCR检测转染后24 h两组miR-125a及IL-23受体（IL-23R）mRNA的表达，采用Western印迹法测定两组转染后48 h IL-23R、Janus激酶2（JAK2）、蛋白激酶B（AKT）和磷酸化AKT（p-AKT）的表达。采用双荧光素酶报告实验验证miR-125a和IL-23R间的靶向关系。两组间均数比较采用t检验，HaCaT细胞增殖能力随时间的变化采用重复测量方差分析法评估。结果 质粒转染后，miR-125a组miR-125a相对表达水平（6.377 ± 0.745）高于miR-NC组（0.700 ± 0.222），差异有统计学意义（t = 7.305，P = 0.002）。转染后0、24、48 h，miR-125a组与miR-NC组细胞增殖能力差异无统计学意义（t值分别为0.663、0.623、1.930，均P > 0.05）；转染后72 h，miR-125a组细胞增殖能力显著低于miR-NC组（t = 4.407，P < 0.05）。MiR-125a组IL-23R mRNA表达水平显著低于miR-NC组（t = 3.082，P < 0.05）。与miR-NC组相比，miR-125a组IL-23R、JAK2和p-AKT蛋白表达量均降低，差异有统计学意义（t值分别为11.715、6.996、12.424，P值分别 < 0.001、 = 0.002、 < 0.001）。双荧光素酶报告实验显示，miR-125a可靶向结合IL-23R。结论 MiR-125a可能通过负性靶向调控IL-23R/JAK2/AKT信号通路抑制角质形成细胞的增殖。

关键词: 屑病, 角蛋白细胞, 微RNAs, 细胞增殖, 白细胞介素23, 微RNA-125a

Abstract: 【Abstract】 Objective To explore the mechanism underlying microRNA （miR）-125a-mediated inhibition of proliferation of keratinocytes. Methods After 24-hour pretreatment with interleukin （IL）-23, human HaCaT keratinocytes were divided into miR-125a group and miR-NC group transfected with a miR-125a overexpression plasmid and a control plasmid, respectively. Cell counting kit-8 （CCK8） assay was performed to evaluate the proliferative ability of HaCaT cells in the two groups at 0, 24, 48 and 72 hours after transfection, real-time fluorescence-based quantitative PCR to determine the mRNA expression of miR-125a and IL-23 receptors （IL-23R） in the two groups 24 hours after transfection, and Western blot analysis to determine the protein expression of IL-23R, Janus kinase 2 （JAK2）, protein kinase B （AKT） and phosphorylated AKT （p-AKT） in the two groups 48 hours after transfection. Dual-luciferase reporter assay was performed to verify the targeting relationship between miR-125a and IL-23R. Comparison of means between two groups was carried out by using t test, and changes in the proliferative ability of HaCaT cells over time were evaluated by using repeated measures analysis of variance. Results After plasmid transfection, the relative expression of miR-125a was significantly higher in the miR-125a group （6.377 ± 0.745） than in the miR-NC group （0.700 ± 0.222; t = 7.305, P = 0.002）. At 0, 24 and 48 hours after transfection, there was no significant difference in cellular proliferative ability between the miR-125a group and the miR-NC group （t = 0.663, 0.623 and 1.930, respectively, all P > 0.05）; at 72 hours after transfection, the cellular proliferative ability was significantly lower in the miR-125a group than in the miR-NC group （t = 4.407, P < 0.05）. The IL-23R mRNA expression was significantly lower in the miR-125a group than in the miR-NC group （t = 3.082, P < 0.05）. Compared with the miR-NC group, the miR-125a group showed significantly decreased protein expression of IL-23R, JAK2 and p-AKT （t = 11.715, 6.996, 12.424, P < 0.001, = 0.002, < 0.001, respectively）. Dual-luciferase reporter assay showed targeted binding of miR-125a to IL-23R. Conclusion MiR-125a may inhibit the proliferation of keratinocytes by negatively regulating the IL-23R/JAK2/AKT signaling pathway.

Key words: Psoriasis, Keratinocytes, MicroRNAs, Cell proliferation, Interleukin-23, MicroRNA-125a