LncRNA-Dleu2协同miR15/16调控CD4+T细胞亚群分化参与多发性硬化发病机制的研究

Multiple sclerosis (MS) is one of the study hotspots in the neuroimmunological diseases and CD4+T helper (Th) cells are considered to play important roles during the pathogenesis of MS. Different type of CD4+ Th cells specific transcriptional factors has been found, it still remains a difficult question to resolve the regulation of these transcriptional factors. In recent years, the great transcriptional regulation function of LncRNA has been recognized. However, it still remains unclear whether LncRNA involves in the pathogenesis fo MS/EAE and regulates the cell specific transcriptional factors to change the distribution of CD4+Th cells. Our previous work demonstrated that LncRNA-Dleu2 involved in the pathogenesis of MS/EAE and probably regulated the differentiation of CD4+Th cells through binding Foxo1. Dleu2 might play coordinate role with miR15a/miR16-1. However we still know little about the function of Dleu2 on the regulation of Th subtypes. Therefore, our study will execute around the role of Dleu2 on the regulation of differentiation of CD4+Th subtypes during the mechanism of MS, and explore the relationship of Dleu2, miR15a/miR16-1 and Foxo1 on the regulation of CD4+Th cell differentiation. Our project will explore new therapies for MS/EAE.

多发性硬化（MS）是神经系统自身免疫性疾病的研究热点，CD4+T细胞亚群失衡被认为在MS发病机制中起重要作用。虽然不同CD4+T细胞特异性转录因子已被发现，但如何调控这些转录因子却一直是个难题。近年来LncRNA强大的转录调控能力被逐渐认识，而LncRNA在MS/EAE发病过程中是否参与了对细胞特异性转录因子的调控从而改变CD4+T细胞亚群分布还不清楚。我们前期筛选初步表明LncRNA-Dleu2参与了MS/EAE的发病过程，并可能通过结合Foxo1从而调控CD4+Th细胞分化，miR15a/miR16-1可能与Dleu2有协同作用。目前我们对Dleu2在Th亚群中的调节作用还所知甚少。为此本项目围绕着Dleu2调控CD4+T细胞亚群分化在MS发病机制中的作用展开，探索Dleu2，miR15a/miR16-1及Foxo1在调控Th细胞亚群分化过程中的关系，为MS/EAE的治疗探索新方法。

经过4年的科研工作，我们如期完成了课题内容。我们通过体内实验，证实了Foxo1/Foxp3/Dleu2/miR15a参与了EAE病程的进展，Dleu2表达的改变能够影响Treg细胞的极化。为深入探究MS/EAE发病过程中lncRNA的调节功能，我们综合RNA-seq与RNA-microarray结果构建了EAE小鼠CD4+T细胞中lncRNA表达文库，通过搭建lncRNA-mRNA互作网络宏观地展现了疾病状态下，lncRNA与mRNA之间复杂的调节关系，并利用生物信息学方法预测了EAE高峰期时差异表达显著lncRNA分子的生物学功能。通过深入挖掘lncRNA表达文库，我们发现在EAE高峰期时差异表达最为显著的lncRNA-NONMMUT031096可能与MS/EAE存在更为密切的关系，在后续研究中针对该lncRNA的作用机制进行了深入研究。体内实验结果显示NONMMUT031096的表达水平在EAE病程中呈动态变化并与EAE病程具有正向相关性。编码能力评测结果证实NONMMUT031096为不具有编码蛋白能力的RNA分子，核质分离实验及细胞特异性检测结果显示NONMMUT031096为主要表达在CD4+T细胞的细胞质中的lncRNA分子，并且该lncRNA的表达仅在EAE病程进展中发生变化。同时结合体外干扰实验，我们发现NONMMUT031096表达降低后，Treg细胞的比例显著升高，lncRNA-mRNA互作网络显示该lncRNA分子与靶向基因Rab27a/Egf的表达密切相关。这说明该lncRNA分子可通过调节调控靶向基因的表达调节Treg细胞的极化，最终参与了EAE的发生发展。我们所取得的成果以科研论文的形式完成文章4篇，其中SCI收录3篇，国内核心期刊1篇，同时培养博士研究生3名，硕士研究生5名，圆满的完成了科研课题。