转录因子SOX5调控类风湿关节炎滑膜成纤维细胞中IL-6表达的分子机制研究

IL-6 plays a critical role in the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RAFLS); however, the mechanism of IL-6 regulation in RAFLS remains unclear. Our previous work had identified a novel transcriptional factor SOX5 which was over expressed in RA synovium, and locally silencing SOX5 expression in the joints of the collagen-induced arthritis (CIA) mouse model could markedly reduce synovitis inflammation and bone erosion. In addition, we had proved that SOX5 was involved in RA bone erosion by mediation of RANKL expression; however, the mechanism of SOX5 in RA inflammation remains unknown. Furthermore, we identified 18 SOX binding sites in the promoter of IL-6 gene by bioinformatics analysis. Based on the above data, we hypothesize that SOX5 plays a critical role in the transcriptional regulation of IL-6 expression in RAFLS. In this study, over-expressed vector, RNA interference, Real-time PCR, Western-blot、luciferase report gene, CHIP and RNA-seq techniques will be used to study the effect of SOX5 on IL-6 expression in vitro and in vivo. Our proposal will not only help to fully understand the molecular mechanism of inflammation in RA, but also facilitate the identification a novel target for RA therapy mediated by regulation IL-6 expression.

IL-6在类风湿关节炎滑膜成纤维细胞（RAFLS）炎症过程中起着重要作用，但其在FLS中表达调控的机制尚不明确。我们前期工作报道了转录因子SOX5在RA滑膜组织中高表达，局部沉默SOX5能显著抑制胶原诱导关节炎鼠滑膜炎症及骨侵蚀，并已证实SOX5通过调控RANKL表达参与RA骨侵蚀，但其介导炎症的机制尚不清楚。通过生物信息学分析我们发现IL-6启动子区有18个SOX转录因子结合位点，因此本研究假说为：SOX5通过调控RAFLS中IL-6的表达参与RA炎症过程。本研究将利用SOX5过表达载体、RNA干扰、PCR、Western、荧光素酶报告基因、CHIP、RNA-seq等技术，从细胞以及动物层面揭示SOX5调控RA-FLS中IL-6表达的分子机制。本研究有助于将深入理解RA慢性炎症机制，并可能从上游揭示RA中特异性调控IL-6表达的分子机制，为RA关节炎症寻找新的干预靶标。

本课题组的前期研究提示转录因子SOX5 极有可能是 RA 炎性滑膜成纤维细胞中特异性调节 IL-6 表达的关键转录因子之一，因此本研究基于假说：SOX5 通过特异性调控 RA 滑膜成纤维细胞中 IL-6 的表达影响 RA 炎症过程，重在阐明在RA的滑膜成纤维细胞中，SOX5 和 IL-6 之间发生的相互作用及相关的分子基础。. 体外实验中，本研究首先通过RNA干扰技术（shRNA-SOX5）和SOX5高表达重组腺病毒载体（Ad-SOX5）等观察了在IL-1β等炎症因子刺激作用下，SOX5对RA滑膜成纤维细胞中 IL-6 表达的影响，结果显示高表达SOX5能够促进IL-6的表达；通过构建 IL-6 启动子区 SOX5 结合位点序列缺失荧光素酶载体，转染滑膜成纤维细胞系MH7A，观察了过表达SOX5后对IL-6启动子转录活性的影响，结果显示过表达SOX5提高了IL-6启动子的转录活性；通过染色质免疫共沉淀技术（ChIP）观察了SOX5调控IL-6转录的反应元件。体内实验中，通过在CIA小鼠模型中沉默局部关节SOX5，观察对局部关节炎症及骨侵蚀的的作用，结果显示抑制局部关节SOX5的表达，能够减轻小鼠局部关节的炎症状态和骨侵蚀的严重程度， Real-time PCR及western-blot观察到滑膜成纤维细胞中IL-6等炎症因子的表达降低。以上实验结果都在一定程度上揭示了SOX5调控IL-6转录的机制。. 本研究是申请人既往原创性研究的延续和深入，进一步揭示了SOX5对炎症通路的影响，有助于深入理解 RA 慢性炎症的病理机制，并从上游转录因子的角度从一定程度上揭示了IL-6调控表达的部分分子机制，为寻找RA新的干预靶标奠定了一定的理论基础。