lncRNA 9194介导BRD4对RA成纤维样滑膜细胞增殖和侵袭的调控作用

Abnormal proliferation and invasion of fibroblast like synoviocytes (FLSs) play a key role in controlling the progress of synovial invasion and joint destruction of rheumatoid arthritis (RA). Our previous studies indicate the critical role of bromodomain protein BRD4 in synovial inflammation, proliferation and invasion, however, the underlying mechanisms of BRD4 inhibition for regulating RA FLSs function is still unclear. In recent years, it has been shown that inhibition of BRD4 suppresses growth and invasion in some kinds of cancer through regulating long noncoding RNA (lncRNA). We have found that expression of lncRNA 9194 is significantly increased in FLSs from RA patients compared with healthy controls. lncRNA 9194 is mainly located in cytoplasm. Inhibition of lncRNA 9194 by specific siRNA suppressed proliferation, migration and invasion of RA FLS. BRD4 inhibition downregulated the expression of lncRNA 9194. Therefore, based on our previous studies, this proposal will be aimed at identifying the role of lncRNA 9194 in regulating proliferation, migration and invasion of RA FLSs. We further search for the “molecular target” that interacts with lncRNA 9194 and regulates FLS function by using bioinformatics analysis, RNA pulldown and RNA immunoprecipitation (IP). We will further determine whether lncRNA is involved in the regulation of BRD4 inhibition in RA FLSs function. We propose to clarify the mechanisms underlying synovial proliferation, invasion and joint destruction of RA in the term of epigenetics, providing the scientific basis for establishment of a novel therapeutic target for RA.

成纤维样滑膜细胞（FLS）异常增殖和侵袭是类风湿关节炎（RA）关节破坏进展的关键因素。我们发现溴区蛋白4（BRD4）在RA患者滑膜病变中发挥关键作用，抑制BRD4能控制RA FLS增殖、迁移和侵袭，明显改善CIA小鼠的关节破坏，但BRD4调控RA FLS功能的分子机制仍不清楚。近期发现BRD4通过长链非编码RNA（lncRNA）调节肿瘤细胞生长和侵袭。利用基因芯片筛选，我们发现lncRNA 9194在RA FLS中表达明显高于正常人，下调lncRNA 9194表达显著抑制FLS增殖、迁移和侵袭；抑制BRD4下调lncRNA 9194表达。本课题拟在上述基础上，深入研究lncRNA 9194对RA FLS增殖和侵袭的调控及其机制，明确lncRNA 9194是否介导BRD4调控RA FLS增殖和侵袭，旨在阐明BRD4调控RA滑膜病变的分子机制，进一步揭示lncRNA在RA发病机制中的作用。

滑膜血管翳的侵袭作用是类风湿关节炎（RA）发生关节破坏的关键因素，成纤维样滑膜细胞（fibroblast like synoviocytes, FLS）是RA滑膜血管翳重要组成细胞，在RA关节软骨和骨质破坏中扮演关键角色。长链非编码RNA（lncRNA）是一类长度大于200 nt的非编码RNA，其在RA等自身免疫疾病的作用远未得到阐明。我们前期发现溴区蛋白BRD4参与调控RA FLS的多种功能，但其是否通过lncRNA发挥作用仍不清楚。本课题运用分子生物学和现代免疫学技术研究发现，lncRNA ENST00000509194（简称lncRNA9194）在RA FLS和滑膜组织中表达增高，功能研究发现lncRNA9194参与调控RA FLS的迁移、侵袭，但对细胞增殖、凋亡和MMP分泌没有调节作用。进一步利用RNA pulldown、质谱分析和生信分析等发现， lncRNA9194与RNA结合蛋白human antigen R(HUR)结合在胞浆形成复合体，进而促进HUR与APPL2 mRNA之间的结合，促进APPL2 mRNA的稳定性，使APPL2蛋白表达增高，从而导致RA FLS增殖和侵袭能力增强。根据其功能，我们将lncRNA9194命名为HAFML（HUR-Associated Fibroblast Migratory LncRNA）。我们还发现抑制BRD4下调HAFML、HUR和APPL2表达，提示HAFML可能介导BRD4调控RA FLS功能的作用。上述研究结果提示滑膜中HAFML表达上调可能是RA发生关节破坏的重要因素，揭示了lncRNA参与RA发病的新机制，为RA关节破坏的治疗提供新的靶点。