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| miR-144 靶向NF-κB受体活化因子配体蛋白调控树突状细胞分泌细胞因子 |
| 郭猛△,刘芳△,宋少华,郑玉廷,张磊,邹游,郭闻渊,丁国善,傅志仁,王正昕* |
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(第二军医大学长征医院器官移植科, 上海 200003
△共同第一作者
*通信作者) |
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| 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-α mRNA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-23 mRNA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-α mRNA的表达不受影响。结论 miR-144 靶向RANKL调控DCs细胞因子分泌。 |
| 关键词: miRNA-144 NF-κB受体活化因子配体蛋白 树突状细胞 细胞因子 |
| DOI：10.3724/SP.J.1008.2014.01053 |
| 投稿时间：2014-02-27修订日期：2014-05-02 |
| 基金项目:国家自然科学基金(81270551)，上海市科委国际合作课题(11410708700). |
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| Regulatory effect of miR-144 on cytokine secretion in dendritic cells by targeting receptor activator of NF-κB ligand |
| GUO Meng△,LIU Fang△,SONG Shao-hua,ZHENG Yu-ting,ZHANG Lei,ZOU You,GUO Wen-yuan,DING Guo-shan,FU Zhi-ren,WANG Zheng-xin* |
(Department of Organ Transplantation, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
△Co-first authors.
*Corresponding authors) |
| Abstract: |
| 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-6 and 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 DC2.4 cell line stably overexpressing the target gene, and detected the TNF-α secretion 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-6 and 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 DC2.4 cells stably overexpressing RANKL resulted in no change of TNF-α mRNA expression. Conclusion miR-144 can regulate DCs cytokine secretion by targeting RANKL. |
| Key words: miRNA-144 receptor activator of NF-κB ligand dendritic cells cytokines |
