rhIL23R-CHR/Fc调控lncRNA靶向Th17治疗自身免疫性疾病的机制研究

IL-23/STAT3 signaling pathway is a key process to Th17 cell differentiation, and Th17 cells have been regarded closely related to the development of autoimmune diseases. We previously designed and prepared a new rhIL23R-CHR/Fc fusion protein for the first time to antagonize endogenous IL-23, which is effective in the treatment of experimental autoimmune encephalomyelitis model in mice. To futher elucidate the mechanism of action, we found that the key transcription factor RORγT in Th17 cells is regulated by lncRNA. Thus, we hypothesize that rhIL23R-CHR/Fc can regulate Th17 cells through lncRNA to balance the immune responses. In this proposal, we will use the techniques of RNAi and CRISPR/Cas9 to dissect the effects of lncRNA on Th17 cell differentiation and on the treatment of EAE model. Meanwhile, we will identify the target gene and its regulation mechanism. Differing from classic approaches to treat autoimmune disease and therapeutic strategies, rhIL23R-CHR/Fc might exert its curative effects through regulating the expression of lncRNA in addition to its function in downstream gene, which will offer new options for the treatment of autoimmune diseases.

IL-23/STAT3信号通路是Th17细胞分化过程中的关键途径，Th17细胞异常被证实与自身免疫性疾病的发病密切相关。本课题组首次设计并制备了新型rhIL23R-CHR/Fc融和蛋白，通过拮抗内源性IL-23有效治疗自身免疫性疾病模型鼠。在深入研究融合蛋白作用机制时，我们发现Th17细胞关键转录因子RORγT的表达受lncRNA调节。因此，我们推测rhIL23R-CHR/Fc通过影响lncRNA来调控Th17细胞所导致的免疫失衡。本研究拟采用RNAi及CRISPR/Cas9技术，验证lncRNA调节Th17细胞的分化功能及对模型鼠治疗的影响；根据与其表达量密切相关的靶基因及信号通路，对lncRNA 发挥作用的分子机制进行探讨。本研究以rhIL23R-CHR/Fc发挥治疗作用为切入点，揭示lncRNA参与药效及调控下游基因的作用模式，为自身免疫性疾病预防、诊断和靶向治疗提供新的思路和策略。

长链非编码RNA是一类具有强大调控功能的调节性RNA分子，相较于microRNA 单一的作用模式，lncRNA具有更为复杂而全面的调控方式网络，可在各个层面影响生物体的生理活动或病理改变。LncRNA在自身免疫性疾病中的作用机制的研究虽有部分报道，但仍有许多功能性lncRNA 及其作用机制等待被发现与阐明。本课题以实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis, EAE）小鼠模型为切入点，利用高通量检测技术获得与EAE 相关的lncRNA表达谱。在此基础上，本课题结合生物信息学分析手段及荧光实时定量PCR技术，并利用工具分子rhIL23R-CHR/Fc筛选获得与EAE及Th17细胞分化相关的lncRNA分子。以慢病毒作为基因转导工具，对目标lncRNA进行过表达或敲减，在Th17细胞极化条件下考察目标lncRNA对Th17细胞分化的影响，并对功能性目标lncRNA分子进行了作用机制探究，结果显示过表达ENSMUST00000174173及干扰ENSMUST00000119414，能够抑制Th17细胞分化，提示 ENSMUST00000174173、ENSMUST00000119414与Th17细胞功能有关。且这两个序列均为首次报道，进行 ENSMUST00000174173和ENSMUST00000119414的亚细胞定位，发现ENSMUST00000174173位于细胞核，ENSMUST00000119414位于细胞质，结合ceRNA分析联合qPCR寻找其相互作用的microRNA分子为miRNA-686，构建lncRNA-microRNA-mRNA互作网络，验证该ceRNA网络对Th17细胞分化与功能具有调控作用。本课题筛选到两个关键lncRNA分子参与自身免疫性疾病进程，并阐明lncRNA在自身免疫性疾病中的作用机理，为自身免疫性疾病提供了新的潜在靶点与标志物。本项目共发表SCI文章四篇，中文期刊2篇。