LncRNA-NONMMUT050830通过调控microRNA182-HIF1α通路参与多发性硬化发病机制的研究

The imbalance of CD4+T cell subsets caused by the abnormal differentiation of pro-inflammatory T cells is the key reason leading to Multiple sclerosis (MS). Researchers found that the differentiation of CD4+T cell subsets are not only controlled by microRNA and related transcriptional factors, but also modulated by lncRNAs which are the key regulation factors of immune system. lncRNAs can function by “sponges” microRNA to inhibit microRNA regulate pathway downstream. Combined high-throughput sequencing database with previous experimental results, we first found and verified that lncRNA-NONMMUT050830 take participate in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The expression of LncRNA-NONMMUT050830 down-regulating in sensitized CD4+T cells, and we also found the LncRNA-NONMMUT050830 has highly conserved binding site with microRNA182. Also, we observe the differentiation of Th1 cells influenced by microRNA182-HIF1α pathway, demonstrate that lncRNA-NONMMUT050830 may regulate the differentiation of Th1 cells by microRNA182-HIF1α pathway, and then destroy the dynamic balance of CD4+T cell subsets, result in MS/EAE finally. With the in-depth study of these new mechanisms, we can deeply understand the important mechanisms of lncRNA-NONMMUT050830 in MS/EAE, even provide theoretical and scientific basis as the clinical guidelines for novel approaches to the treatment of MS.

促炎性T细胞分化异常引发的CD4+T细胞亚型失衡是导致多发性硬化（MS）发病重要原因之一。CD4+T细胞亚型分化不仅受控于microRNA及转录因子，近来发现lncRNA可作为microRNA“海绵”抑制其对下游通路的调控。通过对高通量数据筛选并结合前期工作，我们首次发现并证实lncRNA-NONMMUT050830参与MS动物模型-实验性自身免疫性脑脊髓炎（EAE）的发病进程，在致敏CD4+T细胞中表达下调且与microRNA182有高度保守结合位点。此外microRNA182-HIF1α通路会影响Th1细胞分化，提示LncRNA-NONMMUT050830靶向调控microRNA182-HIF1α影响Th1分化致使 CD4+T细胞亚型失衡，进而导致EAE/MS发生。对这些新机制的深入研究，不仅加深了解lncRNA在EAE/MS发病机制中的重要作用，更为MS新型临床治疗方法提供科学依据。

经过三年的科研工作，我们如期完成了项目内容。通过体内实验证实了lncRNA-NONMMUT050830参与多发性硬化（RRMS）的发病进程。在本研究中，首先检测microRNA 182在复发缓解型多发性硬化症（MS）患者外周血中的表达情况。然后，通过在体内和体外条件下过度表达或沉默microRNA 182的表达来研究microRNA 182在促炎性CD4+T辅助细胞分化中的作用。应用流式细胞术检测CD4+IFN-γ+Th1细胞和CD4+IL-17+Th17细胞的分化情况；使用western blot检测下游HIF-1α转录因子的蛋白表达。结果可见， lncRNA-NONMMUT050830在小鼠体内的表达与疾病的严重程度呈负相关，同时RRMS患者外周抗凝血中的microRNA 182表达上调。在小鼠实验性自身免疫性脑脊髓炎（EAE）模型中，microRNA 182过表达能够加重小鼠的临床症状，同时导致体内CD4+IFN-γ+Th1和CD4+IL-17+Th17分化增强。体外诱导microRNA 182过表达可抑制缺氧诱导因子1α（HIF-1α）的蛋白表达和转录活性，但增加了小鼠CD4+T细胞中IFN-γ转录物的水平；此外，体外抑制microRNA 182的表达能够使HIF-1α表达增加，同时CD4+IFN-γ+Th1和CD4+IL-17+Th17分化被抑制。综上所述，我们的研究结果表明lncRNA-NONMMUT050830参与EAE的发病进程，且与疾病严重程度呈负相关，此过程可能是通过调控microRNA 182的表达来完成的，通过调控下游转录因子HIF-1α从而影响CD4+T细胞分化，总之，microRNA 182-HIF-1α通路可能是调节Th细胞在自身免疫性炎症过程中对自身抗原的反应和抗原特异性Th细胞分化的一种过渡性中枢作用。