微小RNA-155通过抑制Ets-1促进Th17分化  

miR-155 facilitates the differentiation of Th17 cells by inhibiting the gene expression of Ets-1

作　　者：尹志华[1] 罗秀霞[1] 张春容[1] 陈新鹏[1] 黄进贤[1] 叶志中[1] 

机构地区：[1]广东医学院附属深圳福田医院香蜜湖分院、广东医学院风湿病研究所,518040

出　　处：《中华风湿病学杂志》2015年第11期730-734,共5页Chinese Journal of Rheumatology

基　　金：国家自然科学基金（81102266,81301529）;广东省自然科学基金（S2013040012296）;广东省医学科研基金（A2011566）;深圳市科技计划项目（JCYJ20140416094256891）;深圳市福田区科技计划项目（FTWS201308）;深圳市医学重点学科建设资助项目（2005C10）

摘　　要：目的 探讨微小RNA（miR）-155促进Th17分化的作用途径.方法 采用鼠CD4＋T细胞磁珠分离试剂盒分离小鼠脾CD4＋T细胞,加促Th17分化的IL-2、IL-23和IL-6刺激培养后,转染miR-155过表达或抑制慢病毒载体,流式检测CD4＋T细胞IL-17的表达;ELISA检测细胞培养液IL-17分泌水平;荧光定量PCR检测miR-155、IL-17A和Ets-1 mRNA的表达.筛选Ets-1的siRNA干扰序列,检测si-Ets与miR-155共同作用对Th17分化的影响.采用单因素方差分析,两两比较采用Dunnett法,组间比较采用t检验.结果 对照未刺激组几乎不表达和分泌IL-17,对照刺激组表达IL-17的细胞和分泌IL-17增多.4组表达IL-17细胞百分比差异有统计学意义（F=160.549,P＜0.01）,两两比较miR-155过表达组表达IL-17细胞百分比[（39.86±4.62）％]明显高于对照刺激组[（22.02±2.81）％,P＜0.01]和miR-155抑制组[（19.44±1.49）％,P＜0.01];miR-155过表达组上清液中IL-17水平[（1 509±136） pg/ml]也显著高于其他2个组[（923±42） pg/ml,P＜0.01;（767±94） pg/ml] （P＜0.01）.4组上清液中IL-17表达差异有统计学意义（F=260.813,P＜0.01）;两两比较与其他3组相比,miR-155过表达组miR-155高表达（12.53±0.80,1.78±0.14,7.16±0.62,6.47±0.92,P＜0.01）、IL-17A mRNA高表达（46.55±6.71,1.01±0.19,15.62±1.26,14.20±2.73,P＜0.01）,Ets-1 mRNA低表达（0.66±0.10,1.19±0.04,1.01±0.16,1.37±0.27,P＜0.01）.设计了3条针对Ets-1基因的siRNA,其中si-Ets-2抑制效果最好.si-Ets-2或si-Con与miR-155过表达或抑制慢病毒载体进行共转染,si-Ets-2组均比si-Con组IL-17A mRNA表达上调（17.19±3.58和10.08±0.76,t=-3.361,P=0.028）,而Ets-1 mRNA表达下调（0.27±0.01和0.74±0.03,t=-30.275,P＜0.01）,蛋白印迹法检测也可以看出si-Ets-2#组的Ets-1蛋白表达下降,在miR-155过表达组下降更为明显.结论 miR-155可以通过抑制Ets-1的表达而促进CD4＋T细胞向Th17细胞分化.Objective To elucidate the function way of micro RNA（miR）-155 in the differentiation of Th17 cells.Methods CD4＋T cells were separated from mice spleens using MACS CD4＋T cells separatinge kit and cultured with interleukins [interleukin （IL）-2, IL-23 and IL-6] which could induce CD4＋ T cells differentiate into Th17 cells.IL-17 was detected by flow cytometry and enzyme linked immunosorbent assay （ELISA） after transfected with miR-155 mimics or inhibitor lentiviral vectors.The expression levels of miR-155, IL-17A mRNA and Ets-1 mRNA were detected using fluorescent quantitation real-time quantitative polymerase chain reaction （RT-PCR）.The si-Ets and miR-155 co-function for Th17 differentiation was analyzed.Data analysis was perfoemed using one-way analysis of variance （ANOVA） test and Dunnett test for pair-wise comparison and t test.P〈0.05 was considered to be statistically significant.Results The CD4＋T cells were divided into four groups （the untreated control untreat group, the treatment control treat group, the miR-155 mimnics group and miR-155 inhibitor group）.IL-17 was scarcely expressed and secreted in the untreated control untreat group.The cells expression of IL-17 were significantly different among the four groups （F=160.549, P〈0.01）.The cells expressing of IL-17 were higher in the miR-155 mimics group （39.86±4.62）% than those at the miR-155 inhibitor group （22.02±2.81）%, P〈0.01） and in the treated control treat group [（19.44±1.49）%, P〈0.01].The level of IL-17 was also significantly different among the four groups （F=260.813, P〈0.01）.The level of IL-17 was higher in the miR-155 mimics group [（1 509±136） pg/ml] than that in the miR-155 inhibitor group [（923± 42） pg/ml, P〈0.01）;and in the treated control group [（767±94） pg/ml, P〈0.01）.The expression of miR-155 （12.53±0.80 vs 1.78±0.14, 7.16±0.62, 6.47±0.92, P〈0.01） and IL-17A mRNA （46.55±6.71 vs 1.01±0.19,15.62±1.26, 14.20±2.73, P〈0.01） was significantl

关 键 词：CD4阳性T淋巴细胞 白细胞介素17 MIR-155 ETS-1 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]