蛋白精氨酸甲基化转移酶PRMT5调节T细胞亚群分化及其在炎症性肠病中作用的机制研究

Protein arginine methyltransferase 5 is the type II enzymes that catalyze the formation of ω-NG-monomethyl- and symmetrical ω-NG-dimethylargininase, is well known to play important role in transcriptional regulation, signal transduction, tumor genesis. However, the contribution of PRMT5 in the regulation of T cell differentiation and the role on the inflammatory bowel disease remains unclear. In the previous study, we found that PRMT5 expression level is significantly high in the intestinal inflammation mouse model and the inflammatory bowel disease patients. And reducing PRMT5 expression can significantly relieve the severity of the inflammatory bowel disease. But how the PRMT5 regulates the T cells differentiation and the effects of inhibition of PRMT5 in treatment of inflammatory bowel disease needs to be further investigated. Here, we want to reconstitute CD4-cre/PRMT5fl/fl conditional mouse, and measure the effects of PRMT5 in the development of intestinal inflammation in vivo. Next, using RAN-Seq, we will detect the role of PRMT5 in the regulation of epigenetic mechanisms to control T cells differentiation. Furthermore, using the mass spectrum, we will find the proteins that can be methylated by PRMT5, and the contribution in T cells differentiation and inflammatory bowel disease. Also, pharmacological inhibition of PRMT5 to treatment of intestinal inflammation will be used. The innovation of the study is first to explore of the role of PRMT5 in the differentiation of T cells and the inflammatory bowel disease, and illuminate the underline mechanisms. This study will reveal a novel mechanism in which PRMT5 regulates T cells differentiation and provide a new theoretical basis and targets for immunotherapy of inflammatory bowel disease.

PRMT5主要催化蛋白形成单甲基和对称双甲基精氨酸，对转录调节，信号转导，肿瘤发生起重要作用。PRMT5在T细胞亚群分化及炎症性肠病中作用还未有报导。我们前期研究发现实验性结肠炎及炎症性肠病病人中PRMT5表达明显增加，降低PRMT5表达能减轻肠炎严重程度。但PRMT5是如何调控肠炎中T细胞亚群分化及抑制PRMT5对炎症性肠病治疗尚不清楚，本课题拟构建CD4-cre-PMRT5fl/fl条件敲除鼠，体内验证PRMT5在实验性结肠炎中作用；利用RNA-seq检测PRMT5对T细胞亚群分化表观遗传学研究；利用质谱分析肠道组织中蛋白甲基化，明确PRMT5甲基化的靶蛋白及在T细胞分化中作用；利用精氨酸蛋白甲基转移酶抑制剂治疗炎症性肠病。课题首次探索PRMT5在炎症性肠病及对T细胞分化作用并探讨其分子机制。该研究将从PRMT5及T细胞亚群新视点阐明炎症性肠病发病机制，为其免疫治疗提供理论基础。

PRMT5是主要的II型精氨酸甲基转移酶，负责精氨酸残基的对称二甲基化，参与不同的细胞过程，如转录后调控，DNA损伤修复，RNA剪切等。蛋白精氨酸N-甲基转移酶抑制剂1 AMI-1能够增加Treg的数量，功能和减少实验性结肠炎发生。 AMI-1-处理的调节性T细胞过继转移治疗可以减少结肠炎的发生。PRMT5表达在溃疡性结肠炎病人表达增加，该表达被AMI-1处理抑制。此外，PRMT5敲低的T细胞对TGFβ产生更好的反应并促进Tregs分化。 PRMT5通过增强H3K27me3和DNMT1与Foxp3启动子的结合，抑制Tregs分化。此外，PRMT5敲低导致Foxp3启动子区甲基化水平降低。 PRMT5表达与UC患者的Treg成负相关，PRMT5表达的敲低会增加Treg的数量和降低TNFα，IL-6。进一步构建CD4-cre/PRMT5fl/fl条件敲除鼠，利用PRMT5条件敲除鼠，诱导小鼠实验性结肠炎，通过体内实验评估PRMT5在实验性结肠炎中的作用；通过体内研究明确PRMT5对T细胞亚群的分化调节作用；利用单细胞ATAC-seq,RNA-Seq,检测PRMT5对T细胞亚群分化的表观遗传学研究；利用质谱方法分析敲除鼠T细胞的磷酸化和乙酰化情况，以明确PRMT5催化的靶蛋白及其在T细胞亚群分化中的作用；我们的研究阐明了PRMT5调控调节性T细胞功能的新机制，减少PRMT5表达可能成为治疗UC的靶标。