let-7e对PBMCs和原代单核细胞产生IL-10的影响

The effects of let-7e on IL-10 expression in PBMCs and primary monocytes

目的研究let-7e对PBMCs和原代单核细胞产生IL-10的影响。方法生物信息学分析let-7e的靶基因并通过荧光素酶报告实验进行验证;用1μg/ml或10μg/ml LPS刺激PBMCs和原代单核细胞24、48和72 h后,ELISA法检测细胞培养上清中IL-10的质量浓度,筛选出LPS最佳刺激时间和质量浓度;用let-7e mimic或阴性对照(NC)瞬时转染PBMCs和原代单核细胞24、36和48 h后,q RT-PCR和免疫荧光法检测其转染效率;转染细胞经LPS刺激后,ELISA法检测细胞培养上清中IL-10的质量浓度。结果 let-7e可能直接靶向抑制IL-10;1μg/ml LPS刺激细胞24 h即可产生较高水平的IL-10;瞬转let-7e mimic 24 h即可使细胞高表达let-7e;let-7e mimic组细胞培养上清中IL-10的质量浓度低于let-7e NC组。结论 let-7e可抑制PBMCs和原代单核细胞产生IL-10。

The purpose of this study was to evaluate the effect of let-7e on IL-10 expression in the peripheral blood mononuclear cells（PBMCs） and the primary monocytes. Bioinformatics analysis was performed to predict let-7e putative targets and dual-luciferase reporter assay was used to identify the target genes. After stimulation with1 μg/ml or 10 μg/ml LPS for 24 h, 48 h and 72 h, IL-10 in the supernatants of PBMCs and the primary monocytes was quantified by ELISA. PBMCs and the primary monocytes were transfected with let-7e mimic or mimic negative control（NC）, and then q RT-PCR and immunofluorescence assay were used to evaluate the efficiency of transfection.The transfected cells were stimulated with LPS, and then ELISA was used to detect IL-10 concentration in the medium. We found IL-10 was the potential target of let-7e; stimulation with 1 μg/ml LPS for 24 h was optimal to induce IL-10 expression in PBMCs and the primary monocytes; the transfected cells could over-express let-7e significantly after transfection for 24 h; IL-10 concentration was lower in let-7e mimic group than that in let-7e NC group. These findings suggested that let-7e inhibits IL-10 expression in PBMCs and the primary monocytes.