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目的探讨miRNA-144对树突状细胞(dendritic cells,DCs)成熟过程中细胞因子分泌的影响及机制。方法通过对本实验室前期工作中获得的DCs成熟过程中miRNA芯片结果进行数据挖掘,筛选得到DCs成熟过程中显著下调的miRNA(miR)-144,并使用脂多糖(lipopolysaccharide,LPS)刺激体外培养的DCs进行验证;检测miR-144转染DCs后相关细胞因子(TNF-α、IL-1β、IL-6、IL-23)的改变及信号通路(NF-κB、MAPK)活化情况;使用TargetScan预测miR-144的作用靶点并通过双荧光报告系统验证;进一步构建靶蛋白过表达DC2.4细胞系并检测miR-144拟似物转染该细胞系后细胞因子TNF-αmRNA的分泌情况。结果体外培养的DCs经LPS刺激成熟后miR-144的表达下调(P<0.01)。miR-144拟似物转染DCs后,TNF-α、IL-1β、IL-6、IL-23mRNA表达均出现下调(P<0.05,P<0.01),NF-κB磷酸化水平下降。通过信息学分析发现miR-144的潜在靶点为NF-κB受体活化因子配体蛋白基因(RANKL)并通过双荧光报告系统证明了该结论。在RANKL过表达DC2.4细胞系中转染miR-144拟似物后,TNF-αmRNA的表达不受影响。结论 miR-144靶向RANKL调控DCs细胞因子分泌。
Objective To investigate the effect of miRNA-144 on the secretion of cytokines during the maturation of dendritic cells (DCs) and its mechanism. Methods The miRNA (miR -144), which was significantly down-regulated during the maturation of DCs, was screened by data mining of the miRNA microarray in the process of DCs maturation obtained in the previous work in our laboratory. The lipopolysaccharide (LPS) DCs were used to detect the changes of related cytokines (TNF-α, IL-1β, IL-6 and IL-23) and the activation of signal pathways (NF-κB and MAPK) The target of miR-144 was verified by double fluorescent reporter system. The target protein overexpression DC2.4 cell line was further constructed and the secretion of cytokine TNF-αmRNA was detected after transfection of miR-144 mimics. Results The expression of miR-144 in DCs stimulated by LPS was down-regulated (P <0.01). The expression of TNF-α, IL-1β, IL-6 and IL-23 mRNA were down-regulated after miR-144 mimics transfected DCs (P <0.05, P <0.01). The phosphorylation of NF-κB decreased. The potential target of miR-144 was found by informatics analysis to be the NF-κB receptor activator ligand protein gene (RANKL) and this conclusion was demonstrated by a dual fluorescence reporter system. Expression of TNF-a mRNA was not affected after miR-144 mimics were transfected in RANKL over-expressing DC2.4 cell lines. Conclusion miR-144 targets RANKL to regulate cytokine secretion of DCs.