LncRNAHOTAIR/miR-221调控uPAR基因表达在类风湿关节炎成纤维样滑膜细胞增殖和侵袭特性中的作用

Urokinase-type plasminogen activator receptor (uPAR) is a multifunctional receptor on cell surface.Our previous studies report that elevated expression of uPAR markedly affects the proliferation, migration, and invasiveness of fibroblast-like synoviocytes of rheumatoid arthritis (RA-FLSs) and the PI3K/Akt signaling pathway may be responsible for mediating effects of uPAR on FLSs.However, the molecular mechanisms underlying regulation of uPAR overexpression on RA-FLSs are unclear now.We found that miRNA-221 could regulates uPAR expression in RA-FLSs and some malignant cells. By performing bioinformatics analysis,we identified some LncRNAs as targets that may closely related to the physical interaction with miR-221,especially HOTAIR,which contributes to the RA pathogenesis through activation of matrix metalloproteinases in RA synoviocytes. Therefore, we hypothesize that LncRNA HOTAIR may bind to miR-221 and then to promote uPAR expression in RA-FLSs,which affect the tumor-like biologic behaviors of FLSs.In current study, we will utilize FLSs and animal models to investigate the interaction of LncRNA HOTAIR with miRNA-221 and their roles on modulating uPAR expression by using molecular/cellular approaches.

我们前期研究发现，尿激酶型纤溶酶原激活物受体（uPAR）在类风湿关节炎（RA）患者成纤维样滑膜细胞（FLS）中表达增高，能促进FLS增殖、迁移、侵袭，机制上可能与PI3K/AKT通路激活有关，但在RA滑膜中调控uPAR表达的分子机制尚不明确。研究发现miR-221可结合uPAR的3'UTR区而调控uPAR表达，miRNA表达受多重机制调控，LncRNA是其中一种重要调控方式。我们通过生物信息学分析发现数个可能参与miR-221调控的LncRNAs，其中HOTAIR能激活RA-FLS分泌基质金属蛋白酶，造成骨质破坏等。因此我们假设，HOTAIR在RA-FLS中可能通过miR-221调控uPAR表达，参与RA-FLSs异常增殖和侵袭等改变。我们将通过FLS及动物模型，采用CRISPR/Cas9等基因敲低/过表达技术，探讨RA滑膜中HOTAIR调控miR-221而调节uPAR表达的分子机制。

类风湿关节炎（RA）是一种自身免疫性疾病, 以慢性、进展性、破坏性关节病变为主要特征，是我国最常见的一种致残性炎症性关节炎，其中滑膜成纤维细胞在RA病变中其重要作用。本课题组既往研究工作表明uPAR在RA-FLS中高表达，与配体uPAR结合后激活PI3K/AKT通路，促进RA-FLS 细胞增殖、迁移、侵袭，改变细胞周期，抑制FLS 细胞凋亡，但uPAR在类风湿关节炎滑膜中高表达的基因调节机制还不清楚。本项目阐明了uPAR在RA-FLS中的调节机制，与miRNA-221-3p、LncRNA NEAT1_1相关。我们通过qPCR、Western Blot、RNA FISH、腺病毒过表达基因、流式细胞术、Transwell迁移和侵袭实验等技术，我们发现：①在RA-FLS细胞内存在miR-221-3p正向调控uPAR的表达；②通过生物信息学分析、芯片数据筛选以及过表达等实验，验证NEAT1_1通过作为miR-221-3p的分子海绵而发挥对uPAR的调控作用；③在RA-FLS细胞内过表达NEAT1_1后，能显著抑制细胞的迁移、侵袭及炎症因子IL-6的表达，提示NEAT1_1参与RA-FLS细胞“类肿瘤样”生物学特性的调节；④NEAT1_1可能通过miR-221-3p/uPAR轴发挥对RA-FLS细胞的调控作用，在RA发生、发展的过程中起“保护”性作用。我们的研究结果表明，通过调控LncRNA NEAT1_1的表达可能抑制 RA-FLS中 uPAR的高表达，进而调控RA滑膜增生、侵袭和关节破坏等病理进程，从而为 RA的治疗研究提供新思路。