摘要
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 cytokine secretion by dendritic cells（DCs）during their maturation and the related mechanism.Methods miRNA chip results showed markedly down-regulated miRNA-144 during DCs maturation.The miR-144 level was also observed in DCs before and after treatment with lipopolysaccharide（LPS）.The changes of related cytokines（TNF-α,IL-1β,IL-6and IL-23）and the activation of signaling pathway（NF-κB and MAPK）were detected in DCs after the transfection of miR-144.TargetScan was used to predict the target spot of miR-144 and double fluorescence reporting system was used for verification.Furthermore,we established DC2.4cell line stably overexpressing the target gene,and detected the TNF-αsecretion after transfecting miR-144 mimics.Results The miR-144 expression was significantly down-related in DCs stimulated with LPS（P〈0.01）.After the miR-144 mimics were transfected into DCs,expressions of TNF-α,IL-1β,IL-6and IL-23 mRNA were significantly reduced（P〈0.05,P〈0.01）,and NF-κB phosphorylation level was reduced as well.Bioinformatics analysis hinted that receptor activator of NF-κB ligand gene（RNAKL）was the potential target of miR-144,which was also validated by double fluorescence reporting system.Moreover,transfecting miR-144 mimics into DC2.4cells stably overexpressing RANKL resulted in no change of TNF-α mRNA expression.Conclusion miR-144 can regulate DCs cytokine secretion by targeting RANKL.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2014年第10期1053-1059,共7页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(81270551)
上海市科委国际合作课题(11410708700)~~
