NF-κB和miR-103调节环路在类风湿性关节炎中的作用及机制研究

Rheumatoid arthritis is a chronic autoimmune disease characterized by inflammatory-associated injury in synovial joint and the main factor for RA occurrence is the over-expressed inflammatory cytokines and tissue injury mediated by persistent NF-κB activation. MicroRNAs widely participate in the regulation of target genes' expression and play important roles in various diseases. We found by microRNA microarray that miR-103 was downregulated in fibroblast-like synoviocytes in RA patients when compared with normal controls. And our preliminary data showed that downregulation of miR-103 can be mediated by activated NF-κB and that two negative regulatory factors of IκB- - TAK1 and βTrCP are target genes of miR-103, which indicated that NF-κB could be activated by down-regulated miR-103 in RA. So, is there a regulatory circuit between NF-κB and miR-103? Is the circuit critical for over-expression of inflammatory cytokines downstream of NF-κB? In order to clarify these questions, we will investigate the detail mechanism of the mutual regulation and synergism between NF-κB and miR-103, and their effect on the excessive secretion of NF-κB mediated inflammatory cytokines,and on the proliferation and invasion of fibroblast-like synoviocytes. This project will broaden our knowledge on NF-kB signal transduction pathway, and uncover the new mechanism about the amplification of inflammatory responses signals in RA. All these are theory and clinically important for RA.

类风湿性关节炎（RA）是以关节滑膜炎性损伤为特征的慢性自身免疫性疾病。NF-κB持续活化导致炎性因子的过度分泌是RA发生的主要因素。miRNA可广泛参与靶基因表达调控，影响多种疾病进程。我们通过芯片筛选发现，miR-103在RA滑膜细胞中表达显著下调。同时预实验结果表明，miR-103的下调可由NF-κB活化所介导，miR-103又可通过调节TAK1和βTrCP而增强NF-κB活化。那么在RA滑膜细胞中是否存在着NF-κB和miR-103调节环路？该环路是否调节着下游炎性因子的过度分泌及滑膜细胞的功能？为阐明上述问题，本项目拟在前期基础上，深入研究滑膜细胞中NF-κB和miR-103相互调节、协同作用的分子机制及其对NF-κB下游炎性因子分泌以及细胞增殖、侵袭功能的影响。这些研究可拓展对NF-κB信号调控机制的认识，还可发现RA中炎性反应信号放大过程的新机制，具有重要的理论意义和临床意义。

RA是以关节滑膜炎性损伤和骨破坏为特征的慢性自身免疫性疾病。滑膜成纤维细胞（FLS）是关节滑膜的重要组成细胞，在RA关节炎症及软骨破坏中发挥重要作用。持续的滑膜炎症致使FLS细胞增殖/凋亡特性异常、数量急剧增加，继而引起滑膜增生。同时，FLS细胞与关节腔内免疫细胞一起打破了正常关节腔内的免疫平衡，最终导致关节与软骨的损伤。因此，阐明FLS细胞生物学功能变化的调控机制，对揭示RA的发病、致病机制具有重要的指导价值。我们通过芯片筛选发现，miR-103和miR-10a等分子在RA FLS中表达显著下调。随后的研究表明，miR-103和miR-10a可被TNF-α、IL-1β等多种RA经典炎性因子下调，这种下调依赖于NF-κB信号通路的活化，且需要NF-κB下游新合成的转录因子YY1的参与。生物信息学分析、双荧光素酶报告系统检测等分析表明miR-103的靶基因为TAK1、IL-15和DKK1，而miR-10a的靶基因为IRAK4、TAK1和BTRC。其中IRAK4、TAK1和BTRC是NF-κB信号通路活化的重要介导分子，而IL-15和DKK1则是破骨细胞发育分化的调控因子。进一步的研究表明，通过调节靶基因，miR-103a和miR-10a一方面可以协同作用促进NF-κB通路的活化和下游TNF-α、IL-1β、IL-6、IL-8和MCP-1等炎性因子和基质金属蛋白酶MMP-1和MMP-13等的表达，并进而促进FLS细胞的增殖、侵袭和迁移；另一方面可以通过DKK1等因子促进破骨细胞的发育分化，并在MMP分子的协同作用下促进RA的骨破坏。最后，CIA动物实验表明提高miR-103（AgomiR-103a）的水平可以显著改善模型小鼠的临床评分、关节肿胀和关节骨破坏。本项目揭示了RA中存在TNF-α/IL-1β→NF-κB/YY1→miR-10a/miR-103a→NF-κB→TNF-α/IL-1β信号调节环路和TNF-α/IL-1β →NF-κB/YY1→miR-10a/miR-103a→DKK1/IL-15/MMPs→破骨细胞分化信号通路。该研究阐明了TNF-α/IL-1β等炎症因子通过非编码RNA促进RA发生发展的分子机制。拓展了RA中NF-κB信号通路活化及放大的认识，发现了RA中炎性反应和骨破坏的新机制，具有重要的理论意义和临床意义。