微小RNA-148a-3p在寻常型银屑病患者外周血CD4^+T淋巴细胞中的表达及临床意义

Expression of miRNA-148a-3p in CD4^+ T lymphocytes in peripheral blood of patients with psoriasis vulgaris and its clinical significance

目的检测微小RNA(miRNA)-148a-3p在寻常型银屑病患者外周血CD4^+T淋巴细胞中的表达,探讨其在寻常型银屑病发病中的作用。方法2017年7月至2018年4月在广州市皮肤病防治所收集寻常型银屑病患者20例,健康对照20例。抽取受试者外周静脉血,采用免疫磁珠法分选CD4^+T细胞,实时定量PCR检测外周血CD4^+T细胞中miRNA-148a-3p的表达;利用生物信息学软件预测miRNA-148a-3p的潜在靶基因,并通过双萤光素酶报告系统进行验证;采用Western印迹法检测受试者CD4^+T细胞中miRNA-148a-3p潜在靶基因Bim蛋白表达水平。采用SPSS20软件,对于符合正态分布的资料,两个独立样本均数比较采用t检验,对双变量资料计算Pearson相关系数;对不符合正态分布的资料,两样本均数比较采用非参数Mann WhitneyU检验,对双变量资料计算Spearman相关系数。结果寻常型银屑病组18例患者CD4+T细胞中miRNA-148a-3p的表达水平为5.61±1.66,健康对照组12例为1.00±0.26,两组比较,U=12,P<0.05。银屑病患者miRNA-148a-3p表达量与银屑病皮损面积和严重程度指数(PASI)呈正相关(r=0.93,P<0.001)。生物信息学软件预测B淋巴细胞瘤2相互作用的细胞死亡调节因子(Bim)是miRNA-148a的潜在靶基因之一,并通过双萤荧光素酶报告系统得到验证。患者组(11例)CD4^+T细胞Bim表达水平为0.69±0.07,健康对照组8例为0.93±0.06,两组比较,t=4.38,P<0.01。银屑病患者Bim表达量与PASI评分呈负相关(r=-0.774,P<0.01)。结论miRNA-148a-3p在寻常型银屑病患者外周血CD4+T细胞中过度表达,并可能通过调控Bim蛋白的表达,导致CD4+T细胞异常活化,进而参与银屑病的发生、发展。

Objective To determine the expression of miRNA-148a-3p in CD4^+ T lymphocytes in peripheral blood of patients with psoriasis vulgaris,and to explore its role in occurrence of psoriasis vulgaris.Methods Totally,20 patients with psoriasis vulgaris and 20 healthy controls were enrolled from Guangzhou Institute of Dermatology between July 2017 and April 2018.Peripheral venous blood samples were obtained from these subjects,and CD4^+ T lymphocytes were isolated from these peripheral blood samples by magnetic cell sorting system.Real-time quantitative PCR(RT-PCR)was performed to determine the expression of miRNA-148a-3p in CD4^+ T lymphocytes in the peripheral blood.Potential target genes of miRNA-148a were predicted by using bioinformatics software,and verified by using a dual-luciferase reporter system.Western blot analysis was conducted to determine the protein expression of Bcl-2 interacting mediator of cell death(Bim,the potential target gene of miRNA-148a-3p)in the CD4^+ T lymphocytes of the subjects.Statistical analysis was carried out with SPSS 20 software by two sample-t test for comparing the means of normally distributed data,and by Pearson correlation analysis for analyzing the correlation of two variables.If the data were not normally distributed,Mann Whitney U test was used for comparing means between two groups,and Spearman correlation analysis for analyzing the correlation of two variables.Results The miRNA-148a-3p expression in the CD4^+ T lymphocytesin the psoriasis vulgaris group(18 cases,5.61±1.66)was significantly higher than that in the healthy control group(12 cases,1.00±0.26;U=12,P<0.05),and was positively correlated with the psoriasis area severity index(PASI)score(r=0.93,P<0.001).Bim was predicted to be one of the potential target genes of miRNA-148a-3p by bioinformatics software,which was also verified by using a dual-luciferase reporter system.The protein expression of Bim in the CD4^+ T lymphocytes was significantly lower in the psoriasis vulgaris group(11 cases,0.69±0.07)than in the healthy control group(8 cases,0.93±0.06;t=4.38,P<0.01),and the protein expression of Bim in the patients with psoriasis vulgaris was negatively correlated with PASI score(r=-0.774,P<0.01).Conclusion miRNA-148a-3p is overexpressed in CD4^+ T cells in the peripheral blood of patients with psoriasis vulgaris,which may regulate the protein expression of Bim,leading to abnormal activation of CD4^+ T cells,and then participate in the occurrence and development of psoriasis.