FUNDC1通过介导线粒体自噬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

成纤维样滑膜细胞（FLS）异常迁移和侵袭是导致类风湿关节炎（RA）关节破坏的关键，但调控机制不清。我们前期报道能量代谢调控因子PGC1β能促进RA-FLS促炎、迁移和侵袭。预实验表达谱芯片分析发现线粒体自噬调控蛋白FUNDC1在RA-FLS表达升高且能促进其线粒体自噬及ATP产生，促进迁移、侵袭及MMP9表达。因此我们提出假说：RA-FLS高表达的FUNDC1通过介导线粒体自噬引起线粒体功能和细胞能量代谢异常，上调MMP9表达，促进RA-FLS异常迁移和侵袭，从而导致RA关节破坏。本项目拟通过体内外功能实验、蛋白抗体芯片、蛋白质组芯片、免疫共沉淀等技术，明确FUNDC1通过介导线粒体自噬促进RA-FLS迁移和侵袭的调控作用及具体分子机制，并通过CIA关节炎动物模型和RA患者前瞻性随访队列证实FUNDC1对RA关节破坏的关键调控作用及其临床意义，为开发精准治疗RA新药提供新靶点。

Rheumatoid arthritis (RA) is a leading cause of extremity disability in Chinese female adults. Fibroblast-like synoviocytes (FLS) in the lining layer of RA synovium manifest tumor-like properties which can directly invade the adjacent articular cartilage by their excessive migration and invasion with increased production of matrix metalloproteinases (MMPs). However, the underlying regulating mechanism of excessive migration and invasion remains elusive. . Recent studies highlight the significance of immunometabolism in the pathogenesis of RA. We previously reported that the transcription factor PGC1β, a mitochondrial and energy regulatory protein, promoted RA-FLS proinflammatory effect (Arthritis Res Ther, 2014). Recently we found that the elevated PGC1β in RA-FLS can promote their migration and invasion through accelerating oxidative phosphorylation of mitochondria and up-regulating reactive oxygen (ROS) and ATP. To further investigate the role and regulating mechanism of abnormal mitochondria in the excessive migration and invasion of RA-FLS, Arraystar Human mRNA microarray analysis was performed and elevated mitophagy-related genes in RA-FLS including PINK, Parkin, BNIP3 and FUNDC1 were discovered. Further qPCR and Western blot verified the over-expression of FUNDC1 mRNA and protein in RA-FLS and immunocytochemistry staining with MitoTracker Red showed a clear colocalization signal of FUNDC1 and mitochondria in the cytoplasm of RA-FLS. FUNDC1 was first reported as a critical mitochondrial-associated membrane receptor for mitophagy in 2012. Our preliminary data of functional experiments in vitro showed that knockdown of FUNDC1 in RA-FLS can suppress mitophagy and ATP synthesis as well as their migration and invasion by down-regulating MMP9 expression. . Therefore, we hypothesized that elevated FUNDC1 in RA-FLS can promote their migration and invasion by regulating mitophagy, abnormal synthesis of metabolites and up-regulated expression of target genes such as MMP9. . In this project, we firstly perform comprehensive evaluation of mitophagy in RA-FLS under the environment of normal oxygen and hypoxia, including detection of autophagy-related markers, dynamic process of mitophagy by transmission electron microscope, count of mitochondria by MitoTracker Red staining, mitochondrial-associated membrane and mitochondria DNA, and mitochondria function assessment of oxygen consumption rate, levels of ROS and ATP. Secondly, we will perform functional experiments both in vivo and in vitro to clarify the role of FUNDC1 on regulating migration and invasion of RA-FLS, including wound healing assay, trans-well assays for migration and invasion, animal model of severe combined immunodeficiency (SCID) mouse for RA-FLS migration and invasion in vivo. For the mechanism exploration, antibody microarrays, protein microarrays and co-immunoprecipitation will be performed to elucidate the mechanism of FUNDC1 on migration and invasion of RA-FLS and how it modulates MMPs by regulating mitophagy. We also perform in vivo study by collagen-induced arthritis rats with articular injection of lentiviral vector for PGC1β gene silencing or over-expression to confirm the role of FUNDC1 on joint destruction in RA. Finally, in order to clarify the clinical significance of FUNDC1 in RA-FLS, we will detect FUNDC1 expression in the lining FLS of RA synovium by double staining with CD55 and analyze the correlation of baseline FUNDC1 expression in RA-FLS with clinical outcomes especially one-year radiographic progression in a prospective RA cohort.. Our project aims to clarify the molecular mechanism of FUNDC1 on promoting migration and invasion of RA-FLS by regulating mitophagy and synthesis of metabolites. Elevated FUNDC1 in RA-FLS might be a potential therapeutic target for the precise treatment of RA.