lncRNA-RAAT募集PGC1β促进类风湿关节炎成纤维样滑膜细胞迁移和侵袭的机制研究

Migration and invasion of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) are critical for inflammation and joint destruction which cannot be intervened by current therapy. Our previous study showed that peroxisome proliferator-activated receptor gamma coactivator (PGC) 1β, a transcription factor, could promote migration and invasion of RA-FLS as well as the expression of matrix metalloproteinase (MMP) 3 and 9. But how PGC1β achieving the specific location and regulation on target genes remains unknown. Emerging evidence shows that long non-coding RNA (lncRNA) regulates transcription by recruiting transcription factors to the promoter of target genes. Our preliminary experiments showed that elevated lncRNA-RAAT in RA-FLS was positively correlated with joint damage and promoted migration and invasion of RA-FLS. What was more, we identified that lncRNA-RAAT could bind to PGC1β. Therefore, our hypothesis is that elevated lncRNA-RAAT in RA-FLS can promote migration and invasion of RA-FLS by recruiting PGC1β which can up-regulate the expression of target genes such as MMPs. In this project, we plan to perform RA-FLS functional experiments in vivo and in vitro, RNA immune coprecipitation, segments deletion, competitive binding analysis and the correlation analysis of clinical outcomes, in order to elucidate the function and mechanism of lncRNA-RAAT in migration and invasion of RA-FLS, and clarify the clinical significance of lncRNA-RAAT in RA which might be a potential therapeutic target for the precise treatment of RA.

成纤维样滑膜细胞（FLS）迁移和侵袭是类风湿关节炎（RA）关节破坏的关键环节，但现有药物不能阻断。我们前期发现转录因子PGC1β可促进RA-FLS迁移和侵袭及基质金属蛋白酶（MMP）3和9表达，但它如何实现靶基因特异性定位及调控尚不清楚。新近报道长链非编码RNA（lncRNA）可特异性募集转录因子到靶基因启动子区域调控转录。我们预实验发现lncRNA-RAAT在RA-FLS中高表达，与关节破坏程度正相关，促进RA-FLS迁移和侵袭，可与PGC1β结合。由此我们提出假说：高表达的lncRNA-RAAT募集PGC1β上调MMPs等靶基因表达从而促进RA-FLS迁移和侵袭，导致关节破坏。本项目拟进一步采用体内外功能学实验、RNA免疫共沉淀、系列缺失和竞争性结合等技术，阐明lncRNA-RAAT调控RA-FLS迁移和侵袭的作用及机制，通过与临床结局相关分析明确其临床意义，为RA精准治疗提供新靶点。

成纤维样滑膜细胞（FLS）迁移和侵袭是类风湿关节炎（RA）关节破坏的关键环节，但现有药物不能阻断。我们前期预实验发现转录因子过氧化物酶体增殖物激活受体γ辅激活因子（PGC）-1β可促进RA-FLS迁移和侵袭，但其如何实现其靶基因特异性定位及调控尚不清楚。本项目从长链非编码RNA（lncRNA）表观遗传学调控的角度，拟进一步明确RA-FLS高表达的PGC-1β促进RA-FLS迁移和侵袭的机制，结合临床研究分析其与临床结局的相关性，为RA精准治疗提供新靶点。.本项目（1）首先通过横断面分析和前瞻性队列研究，发现RA滑膜高表达的PGC-1β水平与病理滑膜炎、关节破坏程度及1年随访的关机破坏进展正相关，证实滑膜PGC-1β参与RA关节破坏过程。（2）体外构建稳定沉默或过表达PGC-1β的RA-FLS细胞模型，结合细胞功能学实验，明确PGC-1β通过上调MMP-3和MMP-9的分泌促进RA-FLS迁移和侵袭。进一步通过ChIP-seq结合荧光素酶报告基因技术等筛选并验证PGC-1β通过激活ATP5B的转录发挥调控作用。（3）应用Arraystar Human lncRNA芯片高通量筛选出RA-FLS高表达的lncRNA-RAAT并通过核浆分离结合原位杂交技术明确其定位在细胞胞浆。细胞功能学实验证实RA-FLS高表达lncRNA-RAAT并抑制其凋亡，促进其增殖、迁移和侵袭，机制研究发现lncRNA-RAAT可能通过调控Bcl-2/Bax通路抑制RA-FLS凋亡。（4）构建高度免疫缺陷的NCG小鼠建立体内RA-FLS软骨侵袭模型，体内实验发现沉默PGC-1β后ATP5B和lncRNA-RAAT表达下降，RA-FLS的软骨侵袭能力减弱。（5）结合已有的RA患者前瞻性队列进行临床和滑膜病理学研究，提出了基于RA-FLS异常活化的RA滑膜病理新分型并建立了RA不良预后的滑膜病理学预测模型。.本项目按研究计划完成，解决了提出的关键科学问题，达到了研究目标。通过临床研究、体外实验和动物实验阐明了RA-FLS迁移和侵袭的调控机制，并提出了基于RA-FLS异常活化的RA滑膜病理新分型及滑膜病理学不良预后预测模型，为RA精准治疗提供了预测工具和新靶点。本项目资助下已发表SCI论著3篇，核心期刊论著1篇，申请发明专利2项。