GALNT1介导的O-聚糖修饰对类风湿关节炎成纤维样滑膜细胞迁移和侵袭的调控及机制研究

Abnormal migration and invasion of fibroblast-like synoviocytes (FLS) play key roles in joint destruction in rheumatoid arthritis (RA). However, the mechanism contributing to the abnormal biology of RA-FLS is still poor understood. We have found by microarray analysis that mucin-type O-glycan biosynthesis pathway is activated in RA-FLS and the expression of key enzyme GALNT1 is significantly elevated. GALNT1-mediated glycosylation has been proved recently to be involved in the modulation of migration and invasion in several kinds of cancer cells, but the function of GALNT1 in RA remains unknown. We further confirmed the elevated expression of GALNT1 in RA synovium tissue and RA-FLS by immunohistochemistry and immunofluorescence staining, real-time quantitative PCR and Western blot, and found that silencing of GALNT1 suppressed migration and invasion of RA-FLS and decreased phosphorylation of FAK which is the key molecule downstream of integrin family. Integrin family have be reported to be modified by GALNT1 mediated glycosylation. Based on our previous results, we propose that GALNT1 modulates migration and invasion of RA-FLS by mediating O-Glycan modification of target proteins (e.g. integrin), contributing to joint destruction in RA. In this project, we plan to confirm the inhibitory role of GALNT1 on migration and invasion of RA-FLS by in vitro and in vivo migration and invasion assay, to clarify the underlying molecular mechanism through co-immunoprecipitation, mass spectrum and Western Blot, and to prove the modulation of GALNT1 on RA joint destruction through a prospective cohort of RA patients, CIA model and GALNT1 knock-out mice. We aim to clarify the underlying mechanism of joint destruction in RA in term of post-translational modification, providing scientific basis for the establishment of a novel therapeutic target for RA.

成纤维样滑膜细胞(FLS)异常迁移侵袭与类风湿关节炎（RA）关节破坏密切相关。我们基因芯片筛选发现RA-FLS黏蛋白型O-聚糖合成通路异常活化，其关键酶GALNT1表达上调。GALNT1介导的糖基化修饰参与肿瘤迁移侵袭调控，但在RA未见报道。预实验证实RA 滑膜GALNT1表达上调，下调GALNT1表达能抑制RA-FLS迁移和侵袭，并抑制其靶蛋白integrin家族下游关键蛋白FAK磷酸化。我们推测：GALNT1可能通过介导integrin等靶蛋白糖基化修饰调控RA-FLS迁移和侵袭参与RA关节破坏。本项目拟进一步通过体内外功能实验明确GALNT1对RA-FLS迁移和侵袭的调控，并通过免疫共沉淀、质谱检测等阐明调控分子机制，通过RA患者前瞻性队列、CIA模型及GALNT1敲除小鼠证实GALNT1对关节破坏的调控，旨在从蛋白质糖基化修饰的角度阐明RA关节破坏发病机制，为RA治疗提供新靶点。

类风湿关节炎（Rheumatoid Arthritis，RA）是常见的自身免疫性疾病，致残率高，目前尚无治愈手段。RA成纤维样滑膜细胞（fibroblast-like synoviocytes，RA-FLS）是RA关节破坏的关键致病细胞。RA-FLS异常迁移和侵袭的特性是导致RA关节破坏的重要因素，但是其调控机制仍不明确，是当前RA研究领域的重点和难点。本项目研究结果证实RA滑膜组织中GALNT1表达明显升高。通过体外细胞实验和CIA关节炎动物模型实验证实GALNT1可能通过调节NEK9分子发挥调控RA-FLS迁移、侵袭的作用。本项目从糖基化修饰的角度出发，提出了一种新的调控RA-FLS迁移和侵袭的分子机制，首次提出GALNT1可能通过调节NEK9糖基化调控RA-FLS迁移和侵袭，为RA发病机制研究提供了的新的思路，也为RA的治疗提供了新的潜在靶点。

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