MiRNA-203在寻常性银屑病皮损的表达及其对HaCaT细胞增殖的影响

目的 研究微小核糖核酸(miRNA)-203在寻常性银屑病患者皮损中的表达,并探讨对角质形成细胞株(HaCaT细胞)增殖的影响.方法 取2014-2016年23例寻常性银屑病患者的皮损组织和相邻非皮损组织.荧光定量PCR法检测组织中miRNA-203的表达水平,并以5'端、3'端地高辛标记的探针对皮肤组织切片中目的miRNA进行原位杂交,观察miRNA-203在皮肤组织中的定位情况.将miRNA-203模拟物(miRNA-203模拟物组)和miRNA-203模拟物阴性对照(阴性对照组)分别转染HaCaT细胞,正常细胞培养组作为空白对照组,采用噻唑蓝(MTT)法、流式细胞仪和Western印迹法分别对HaCaT细胞的增殖、细胞周期及相关周期蛋白(Cyclin D1、Cyclin B1)的变化进行检测.结果 miRNA-203特异性地表达在表皮的角质形成细胞中,除细胞核外,细胞质亦有表达,且寻常性银屑病患者皮损组织中miRNA-203表达水平(1.35±0.28)显著高于非皮损组织(0.52±0.09),差异有统计学意义(t=6.76,P=0.012).转染miRNA-203模拟物能抑制HaCaT细胞增殖(F=9.36,P=0.007),且空白对照组、阴性对照组和miRNA-203模拟物组HaCaT细胞增殖率均随时间的延长逐渐增加(F=18.68,P＜0.001).与阴性对照组和空白对照组相比,miRNA-203模拟物组HaCaT细胞被阻滞在G2/M期(G2/M期细胞比例:31.33％±4.56％比17.02％±3.53％、16.67％±3.32％,均P＜0.05),HaCaT细胞周期蛋白周期蛋白D1表达水平较高(1.15±0.13比0.52±0.05、0.56±0.07,均P＜0.05),而周期蛋白B1水平较低(0.43±0.08比0.93±0.16、0.91±0.0.15,均P＜0.05).结论 miRNA-203可能参与了寻常性银屑病的发生发展过程.

Expression of miRNA-203 in psoriasis vulgaris skin lesions and its effect on the proliferation of HaCaT cells

Objective To investigate the expression of miRNA-203 in skin lesions of patients with psoriasis vulgaris,and to explore its effect on the proliferation of a human keratinocyte cell line HaCaT.Methods Lesional skin and adjacent non-lesional skin tissues were obtained from 23 patients with psoriasis vulgaris from 2014 to 2016.Fluorescence-based quantitative PCR was performed to determine the expression of miRNA-203 in these skin tissues.Targeted miRNA in skin tissues was in situ hybridized by using 5'and 3'digoxigenin-labelled probes,so as to localize the expression of miRNA-203 in skin tissues.Cultured HaCaT cells were divided into 3 groups:miRNA-203 mimic group and negative control group transfected with miRNA-203 mimics and negative control miRNA-203 respectively,and blank control group receiving no treatment.Methyl thiazolyl tetrazolium (MTT) assay,flow cytometry and Western blot analysis were performed to investigate changes in cellular proliferative activity,cell cycle and its related proteins Cyclin D1 and Cyclin B1 in HaCaT cells respectively.Results MiRNA-203 was specifically expressed in epidermal keratinocytes.Besides the cell nuclei,it could be expressed in the cytoplasm.In the patients with psoriasis vulgaris,the expression of miRNA-203 was significantly higher in lesional skin tissues than in non-lesional skin tissues (1.35 ± 0.28 vs.0.52 ± 0.09,t =6.76,P =0.012).The transfection with miRNA-203 mimics could significantly inhibit the proliferation of HaCaT cells (F =9.36,P =0.007).Additionally,the blank control group,negative control group and miRNA-203 mimic group all showed a gradual increase in proliferative activity of HaCaT cells over time (F =18.68,P ＜ 0.001).HaCaT cells were arrested in G2/M phase in the miRNA-203 mimic group with the percentage of cells in G2/M phase being 31.33％ ± 4.56％,compared to 17.02％ ± 3.53％ in the negative control group (P ＜ 0.05) and 16.67％ ± 3.32％ in the blank control group (P ＜ 0.05).Moreover,the miRNA-203 mimic group showed significantly higher protein expression of Cyclin D1 (1.15 ± 0.13),but significantly lower protein expression of Cyclin B1 (0.43 ± 0.08),compared with the negative control group (0.52 ± 0.05,0.93 ± 0.16,respectively,both P ＜ 0.05) and blank control group (0.56 ± 0.07,0.91 ± 0.0.15,respectively,both P ＜ 0.05).Conclusion MiRNA-203 may participate in the occurrence and development of psoriasis vulgaris.