Su Xian Wei, Lu Gang, Leung Chi Kwan, Liu Qiang, Li Yi, Tsang Kam Sze, Zhao Shi Dou, Chan Danny Tat Ming, Kung Hsiang Fu, Poon Wai Sang
Division of Neurosurgery, Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China.
Center for Reproductive Medicine, Shandong University, Jinan, China.
Cell Prolif. 2017 Oct;50(5). doi: 10.1111/cpr.12358. Epub 2017 Jul 21.
MicroRNAs (miRNAs) are considered as the cellular regulators which post-transcriptionally modulate gene expression in diverse biological processes including cell development and immunity. In this study, we investigated functions of miR-181d in dendritic cells (DCs) maturation, and the underlying mechanisms were also explored.
Here we did the miRNA screening in human DCs in response to lipopolysaccharides (LPS) by quantitative real-time PCR (qRT-PCR). The expressions of DCs maturation markers were measured after miRNA mimics transfections. The pharmacological inhibitors of signalling pathways were applied to examine miR-181d effect on DCs maturation by Western blot. Luciferase assay and mixed lymphocyte reaction (MLR) were also performed to reveal the target gene of miR-181d and test the viability of T cells treated with miR-181d transfected DCs.
Overexpression of miR-181d per se is sufficient to promote DCs maturation, and up-regulate CD80 and CD83 expressions without LPS. Besides, we showed that miR-181d activated NF-κB pathway and also promoted the expression of pro-inflammatory cytokine IL12 and TNF-α. Inhibition of NF-κB pathway suppressed DCs maturation. Luciferase reporter assay and target gene knockdown assay indicated that miR-181d targets regulator cylindromatosis (CYLD), a primary negative regulator of NF-κB pathway. MLR assay showed that miR-181d-transfected DCs could promote T-cell proliferation than iDCs in vitro.
Our study demonstrates that miR-181d is required for DCs maturation through the activation of NF-κB pathway by targeting CYLD.
微小RNA（miRNA）被认为是细胞调节因子，可在转录后调节包括细胞发育和免疫在内的多种生物学过程中的基因表达。在本研究中，我们研究了miR-181d在树突状细胞（DC）成熟中的功能，并探讨了其潜在机制。
我们通过定量实时PCR（qRT-PCR）对人DCs中响应脂多糖（LPS）的miRNA进行筛选。在转染miRNA模拟物后测量DC成熟标志物的表达。应用信号通路的药理学抑制剂通过蛋白质印迹法检测miR-181d对DC成熟的影响。还进行了荧光素酶测定和混合淋巴细胞反应（MLR）以揭示miR-181d的靶基因并测试用miR-181d转染的DC处理的T细胞的活力。
miR-181d的过表达本身足以促进DC成熟，并且在没有LPS的情况下上调CD80和CD83的表达。此外，我们表明miR-181d激活了NF-κB通路，还促进了促炎细胞因子IL12和TNF-α的表达。抑制NF-κB通路可抑制DC成熟。荧光素酶报告基因测定和靶基因敲低测定表明miR-181d靶向调节因子圆柱瘤蛋白（CYLD），它是NF-κB通路的主要负调节因子。MLR测定表明，在体外，miR-181d转染的DC比未成熟DC更能促进T细胞增殖。
我们的研究表明，miR-181d通过靶向CYLD激活NF-κB通路来促进DC成熟。
