Mechanisms underlying microRNA-125a-mediated inhibition of proliferation of HaCaT cells by targeting the interleukin 23 receptor signaling pathway: a preliminary study / 中华皮肤科杂志

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.