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

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)24h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 H...

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

【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.