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

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 realtime 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. Key words: CD4-positive T lymphocytes; Interleukin-17; miR-155; Ets-1