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。 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 targe
机构地区 沈阳市第七人民医院皮肤科 空军特色医学中心皮肤科
出处 《中华皮肤科杂志》 CAS CSCD 北大核心 2021年第6期499-503,共5页 Chinese Journal of Dermatology
基金 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)。
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 Psoriasis Keratinocytes MicroRNAs Cell proliferation Interleukin-23 MicroRNA-125a
分类号 R73 [医药卫生—肿瘤]
