miR-155 facilitates the differentiation of Th17 cells by inhibiting the gene expression of Ets-1 / 中华风湿病学杂志

ABSTRACT

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 significantly higher than that in the other three groups, while the expression of Ets-1 mRNA was significantly lower (0.66±0.10 vs 1.19±0.04, 1.01±0.16, 1.37±0.27, P＜0.01).si-Ets-2 was screened because it markedly inhibited the expression of Ets-1 mRNA among the three designed siRNAs.The expression of IL-17A mRNA was higher (17.19±3.58 vs 10.08±0.76, t=-3.361, P=0.028) and the expression of Ets-1 mRNA was lower (0.27±0.01 vs 0.74±0.03, t=-30.275, P＜0.01) in si-Ets-2 group than that in si-Con group when si-Ets-2 or si-Con was co-transfected with miR-155 mimics or inhibitor lentiviral vectors.The expression of Ets-1 protein was lower in si-Ets-2 group than that in si-Con group by Western blotting and the decrease was markedly obvious in the miR-155 mimics group.Conclusion miR-155 can induce CD4+T cells to differentiate into Th17 cells by inhibiting the gene expression of Ets-1.