miR-223调控mTOR/S6K通路促进类风湿关节炎滑膜成纤维细胞增殖的分子机制研究

The proliferation of synoviocytes in rheumatoid arthritis (RA) is one of key factors which result in the erosion of bones. However, the mechanism for those cells to proliferate is not very clear till now. The expression level of miR-223 in synoviocytes was elevated significantly, meanwhile, miR-223 can promote the proliferation and inhibit apoptosis of synoviocytes in vitro in our preliminary work and others’ work. We found that S6K was a direct target gene of miR-223 by bioinformatic prediction, meanwhile, we also found the expression levels of S6K and mTOR in synoviocytes were elevated significantly, and there was a negative correlation between the levels of miR-223 and S6K mRNA in our preliminary work. . Based on our results and prediction, we came up with the following hypothesis: miR-223 activates mTOR/S6K pathway and promotes the proliferation and secretion of synoviocytes by regulating the expression of S6K. In order to get it, we will do it in whole and in vitro. We will promote and inhibit the expression of miR-223 in synoviotes by transfecting miR-223 mimics and inhibitors with cationic liposomes, detect S6K by dual-luciferase reporter gene assay, and activate and inhibit the target gene of miR-223, S6K, in mTOR pathway to observe and verify that miR-223 has a direct effect on the expression of S6K, and promote the proliferation and secretion of synoviocytes. Our research will contribute to clarify the pathogenesis of RA, and propose the theoretical foundation for RA therapy.

类风湿关节炎（rheumatoid arthritis, RA）滑膜成纤维细胞增殖是导致其骨侵蚀的关键因素之一，但滑膜成纤维细胞增殖的机制仍不明确。我们前期及其他课题组研究发现miR-223在RA滑膜成纤维细胞中表达明显升高、体外有促进细胞增殖和抑制凋亡的作用。我们生物信息学预测表明S6K是miR-223作用的一个直接靶点，而且前期发现RA滑膜细胞mTOR、S6K表达明显升高， miR-223与S6K mRNA表达负相关。因此我们提出miR-223通过调控mTOR/S6K信号通路，促进RA滑膜成纤维细胞增殖、分泌的假说。本研究将从整体和离体研究两个层面，采用miR-223过表达与沉默、信号通路靶点激动与抑制、双荧光素酶报告基因检测等手段，验证miR-223调控mTOR/S6K通路对滑膜成纤维细胞增殖、分泌的影响。本研究将有助于阐明RA的骨侵蚀机制，为RA的靶向治疗提供理论依据。

类风湿关节炎（Rheumatoid Arthritis, RA）的主要病理表现为受累关节的滑膜细胞类似肿瘤样高度增殖，增殖细胞分泌的大量炎性介质（如TNF-a、IL-6、IL-1、MMP3 等）与关节骨侵蚀密切相关。因此找到导致滑膜细胞增殖的关键信号通路及信号分子对RA 的靶向治疗至关重要。哺乳动物雷帕霉素靶点（the mammalian target of rapamycin, mTOR）可感受和汇聚细胞外的各种信号（如营养、能量、生长因子、压力等），导致mTOR 通路及其下游分子S6K 被活化和磷酸化，S6K 再磷酸化其靶分子核糖核蛋白S6（RPS6），从而促进各种生物合成过程，包括蛋白合成、细胞的增殖、细胞生长等。文献报道miR-223在RA患者关节滑液和滑膜组织中表达较OA和正常对照明显升高，miR-223可促进破骨细胞的分化和功能。本次研究我们利用生物信息学软件TargetScan预测S6K是miR-223调控的一个直接靶基因。因此滑膜细胞中miR-223可能通过调控S6K表达影响滑膜增殖，参与RA的骨侵蚀。.本次研究证实了miR-223在RA滑膜组织中的表达较正常对照明显升高，S6K mRNA表达水平在RA组滑膜组织中较正常对照组明显降低，并且在RA滑膜组织中miR-223表达水平与S6K mRNA表达水平呈负相关；RPS6 mRNA在RA组滑膜组织中表达较正常对照明显降低,并且在RA滑膜组织中miR-223表达水平与RPS6 mRNA表达水平呈负相关，上述结果表明在RA患者滑膜细胞中miR-223可能调控S6K从而导致S6K及S6K下游分子RPS6表达下降。S6K上游信号分子mTOR mRNA在RA组和正常对照组滑膜组织中表达水平差异无统计学意义，并且在RA滑膜组织中miR-223表达水平与mTOR mRNA表达水平无明显相关性，上述研究结果表明在RA患者滑膜细胞中miR-223可能只作用于S6K而不作用于S6K上游的分子mTOR。.上述研究结果为RA骨侵蚀的发生机制指明了进一步研究的方向，为RA的靶向治疗提供了理论依据。