NUTM2A-AS1通过miR-17和miR-206调控TIMP1/3的表达影响RA-FLS迁移、侵袭及软骨侵蚀机制的研究

Our previous studies showed that the expression of NUTM2A-AS1, TIMP1 and TIMP3 in RA-FLS were significantly down-regulated, while miR-17 and miR-206 were significantly up-regulated. Further studies demonstrated that overexpression of NUTM2A-AS1, TIMP1 or TIMP3 in RA-FLS obviously decreased the migration and invasion, and overexpression of miR-17 or miR-206 in RA-FLS enhanced the cellular migratory and invasive ability. Bioinformatics analysis demonstrated that NUTM2A-AS1 could regulate the expression of TIMP1 and TIMP3 via miR-17 and miR-206, respectively. Based on the previous work, we will perform gene overexpression, gene knockdown or knockout to conduct a series of researches, in which the joint synovial tissues in patients with RA, primary cultured RA-FLS, RA-FLS cell strain and SCID-HuRAg mouse model will be taken as the research objects. We aim to clarify the mechanism of NUTM2A-AS1 regulating the expression of TIMP1/3 via miR-17 and miR-206 respectively to affect the migration and invasion of RA-FLS, and further contribute to the erosion and damage of cartilage. This study may provide theoretical basis and novel targets for the development of the treatment measures of RA.

前期研究发现，RA-FLS中的NUTM2A-AS1、TIMP1和TIMP3表达显著下调，而miR-17和miR-206明显上调；过表达NUTM2A-AS1、TIMP1或TIMP3的RA-FLS迁移和侵袭能力明显下降，而过表达miR-17或miR-206的RA-FLS迁移和侵袭能力明显增强。生物信息学方法关联分析发现，NUTM2A-AS1可能通过miR-17和miR-206分别调控TIMP1和TIMP3的表达。本课题拟在前期工作基础上，采用基因过表达、干扰表达或敲除等方法，以RA患者关节滑膜组织、原代培养的RA-FLS和RA-FLS细胞株以及SCID-HuRAg小鼠模型为研究对象，阐明NUTM2A-AS1通过miR-17和miR-206分别调控TIMP1和TIMP3的表达影响RA-FLS的迁移和侵袭，进一步导致软骨的侵蚀和破坏的机制。本研究可能为发展相应的治疗RA措施提供理论依据和新靶标。

我们的前期工作发现在RA-FLS中NUTM2A-AS1、TIMP1和TIMP3表达显著下调，而miR-17和miR-206明显上调；过表达NUTM2A-AS1、TIMP1或TIMP3的RA-FLS迁移和侵袭能力明显下降，而过表达miR-17或miR-206的RA-FLS迁移和侵袭能力明显增强。生信学方法关联分析显示，NUTM2A-AS1可能通过miR-17和miR-206分别调控TIMP1和TIMP3的表达。为了探讨NUTM2A-AS1调控RA-FLS迁移和侵袭的机制，课题组从RA患者关节滑膜组织、SCID-HuRAg动物模型以及体外细胞水平开展了研究。课题组首先通过体内组织水平相关基因表达水平的检测，证实了在RA患者关节滑膜组织中NUTM2A-AS1表达下调、miR-206、miR-17-5p表达上调，而TIMP1和TIMP3的表达下调。进一步通过建立SCID-HuRAg动物模型证实了NUTM2A-AS1可调控RA-FLS对关节软骨的侵蚀。其次，本课题通过采用腺（慢）病毒介导的基因过表达或干扰表达的方法，在体外细胞水平进行了实验研究，明确了NUTM2A-AS1、miR-206和miR-17-5p在RA-FLS迁移、侵袭过程中发挥了重要的调控作用，初步揭示了miR-206可通过靶向TIMP3负调控TIMP3的表达，从而抑制RA-FLS中MMP2、MMP9的分泌，进一步影响RA-FLS的迁移与侵袭。研究结果还表明，miR-17-5p有可能通过调控METTL14和 YTHDC2的表达，增加RA-FLS中RNA m6A 修饰水平，进而调控EMT相关基因的表达水平，影响RA-FLS 的迁移和侵袭（这方面的机制有待进一步完善）。进一步研究表明NUTM2A-AS1可能并非通过竞争结合miR-206和miR-17-5p而调控TIMP1或TIMP3表达影响RA-FLS的迁移和侵袭，而是通过与具有组蛋白去甲基化酶活性的RIOX2相互作用而影响 RA-FLS中H3K9me2和H3K4me3的水平，从而调控细胞中与上皮间质转化（EMT）相关的基因表达，影响RA-FLS的迁移和侵袭。本研究不仅有助于完善我们对LncRNA UNTM2A-AS1、miR-206和miR-17-5p等参与RA-FLS迁移和侵袭过程调控机制的理解，也有可能为发展相应的治疗RA措提供理论依据和新靶标。