MicroRNAs对缺氧环境中RA关节滑膜成纤维细胞的调控及其机制

Increasing evidence suggests that tissue hypoxia, one the major characteristics of rheumatoid arthritis(RA), maintains the synovial inflammation. Recent studies have established that microRNAs (hypoxamiR), induced by hypoxia in (hypoxia-induciblefactor 1, HIF-1)-dependent or independent pathway, is a feature of the hypoxic response. However, whether hypoxamiR play a role in the regulation of biological characteristics of fibroblast—like synoviocytes（FLS） under hypoxia is still remain to be elucidated. Our previous study found that the expression of IL-1β, IL-6 and IL-8 was markedly enhanced in FLS that were cultured under hypoxia. In this study, miRNA microarray technology is to be used to analyze the differential expression of miRNAs in the oxidative stressed and normal cultured FLS, and then Real Time PCR is used to verify the observation. Further, miRNA mimics and inhibitors will be transfected into RA-FLS, then the proliferation, apoptosis, invasion of FLS,expression of HIF-1α as well as its cytokines secretion including VEGF，IL-1β, IL-6, IL-8 are accordingly detected to determine in the regulation of biological characteristics of FLS by miRNAs under hypoxia. The molecular mechanism including the miRNAs induction signaling pathway and its downstream target gene will also be investigated. This study will clarify the regulation of miRNAs on the FLS and molecular mechanisms and it will develop a novel therapeutic strategy against RA.

类风湿性关节炎(RA)患者关节腔内存在缺氧情况，推动着滑膜炎症持续进展。细胞在缺氧条件下产生大量特征性的microRNAs（miRNAs）,也被称为hypoxamiR，是低氧环境中参与适应性应答的重要成员。而hypoxamiR是否参与了缺氧环境下滑膜成纤维样细胞（FLS）的一系列生物学行为的调控仍有待研究。本项目前期研究证实缺氧可促进FLS增殖并分泌大量炎性细胞因子。在后续研究中，本课题组将应用高通量技术筛选出一组缺氧诱导FLS的差异表达miRNAs。通过过表达和抑制实验研究候选miRNAs对FLS缺氧应答的影响，包括其增殖凋亡水平，侵袭性，表达HIF-1α的水平及VEGF和炎性细胞因子的分泌，并进一步研究miRNAs调控FLS的作用机制，包括其诱导机制及其下游靶蛋白。目的是阐明miRNAs对缺氧环境中RA关节滑膜成纤维细胞的调控及其机制，以期为类风湿性关节炎的治疗找到新的分子靶标。

类风湿关节炎（rheumatoid arthritis, RA）患者关节滑膜衬里层的成纤维样滑膜细胞 (fibroblast like synoviocytes ,FLS) 异常增殖，是RA关节滑膜增生和破坏的关键环节。由于RA-FLS类肿瘤样的增殖和大量炎症细胞的浸润，导致局部毛细血管氧弥散距离增加和耗氧量增加，致RA关节腔内缺氧。研究发现肿瘤细胞等在缺氧条件下产生大量特征性的microRNAs（miRNAs）,也被称为hypoxiamiR，是低氧环境中参与适应性应答的重要成员。本课题组分离培养原代RA-FLS细胞，并将RA-FLS分别置于缺氧环境（3%O2）和常氧环境（21%O2）中培养。应用高通量技术筛选出一组缺氧环境中RA-FLS的差异表达miRNAs。 其中miR-191在三株缺氧培养的RA-FLS中均显著上调表达。进一步研究发现过表达miR-191可以缩短RA-FLS细胞周期，促进RA-FLS增殖；而抑制miR-191可延长RA-FLS细胞周期，抑制RA-FLS增殖。拮抗miR-191后，RA-FLS细胞抗凋亡蛋白BCL-2及细胞周期蛋白CyclinD1均下调表达，并且凋亡蛋白caspase-3的活化增多。靶基因筛选中发现，3’ UTR荧光报告结果显示，miR-191可与C/EBPβ 3’UTR区域结合，证实C/EBPβ为miR-191的靶基因；过表达miR-191显著下调CCAAT/增强子结合蛋白β（C/EBPβ）的mRNA和蛋白水平的表达。敲除C/EBPβ可模拟miR-191的作用缩短RA-FLS周期而促进细胞增殖。目的是阐明miR-191诱导的RA-FLS增生的分子机制，以期为类风湿关节炎的治疗寻找新的分子靶标。