m6A修饰与系统性红斑狼疮发病的相关性研究

Abnormal T cell function is closely related to the initiation and progression of systemic lupus erythematosus (SLE). Recent studies have shown that m6A modification can modulate gene expression through its effect on pre-mRNA splicing, the stability and translation of message RNA (mRNA), and so on. m6A modification can participate in immune response, and its anomaly is implicated in tumorigenesis. Therefore, our research group hypothesizes that abnormal m6A modification may impact RNA stability, translation, etc., leading to abnormal expression of certain important genes and dysfunction of T cells and thereby contributing to the etiology of SLE. By virtue of case-control study, we plan to collect T cells from SLE patents and healthy controls and then measure the m6A level of total RNA and the expression of enzymes involved in m6A modification. As for the differentially expressed enzyme, we will knockdown and overexpress it in T cells and analyze the influence of its knockdown and overexpression on apoptosis and cytokine secretion of T cells. At the same time, we will also collect peripheral blood mononuclear cell (PBMC) from SLE patents and healthy controls and screen those mRNAs which are both differentially m6A modificated and differentially expressed between the two groups. We will further validate the differential m6A modification and expression of some crucial genes for T cells among the aforementioned mRNAs. We will then construct cell lines with stable METTL3 knockdown or FTO knockdown and determine the influence of their knockdown on the mRNA level, protein abundance, and stability of these crucial T cell genes. Accomplishing this project will help reveal the role of m6A modification in the pathogenesis of SLE and provide clues to the identification of new SLE therapeutic targets.

T细胞功能异常与系统性红斑狼疮（SLE）发生与发展密切相关。近期研究表明m6A修饰影响mRNA剪接、稳定性和翻译等方面调控基因表达，可参与免疫应答，其异常与肿瘤发生有关。课题组假设：m6A修饰异常可能通过影响RNA稳定性和翻译等，引起重要T细胞相关基因表达异常和T细胞功能紊乱，参与SLE发病。本课题拟采取病例对照设计，收集SLE患者和健康对照者T细胞，检测总RNA m6A水平和m6A修饰酶的表达，针对异常表达的修饰酶，实现其在T细胞内的干扰和过表达，检测干扰和过表达对凋亡和细胞因子分泌的影响。同时收集SLE患者和健康对照者PBMC细胞，筛选m6A修饰和表达均异常的mRNA，对其中的重要T细胞相关基因进行验证，分别构建稳定干扰METTL3和FTO的细胞系，分析干扰对其mRNA水平、稳定性和蛋白水平的影响。本项目的完成有助于揭示m6A修饰在SLE发病机制中的作用，为发现新的治疗靶点提供线索。

m6A修饰在疾病发生、发展，乃至疾病治疗和预后都有可能扮演着重要角色，有望成为治疗和预防疾病的新干预靶点和生物标志物。然而m6A修饰在SLE中的研究仍十分有限，需进一步的研究。本研究借助MeRIP-seq技术建立SLE疾病mRNA的m6A修饰谱和表达谱，同时筛选已经报道的m6A甲基化酶。结果发现与对照相比，SLE疾病中m6A甲基化水平降低。m6A修饰peaks峰主要集中在终止密码子附近，同时在SLE病例中发现了经典的甲基化结合位点Motifs。研究初步揭示了m6A修饰参与SLE疾病过程。联合分析m6A测序和转录组测序发现，SLE病例组和对照组中的m6A修饰peak（FDR<0.05）分别分布于8052和7225个基因中，其中有6293个基因在两组中都检测到peak。peak主要分布于3’UTR和CDS交界处，即终止密码子附近。利用HOMER(4.1)对peak进行motif预测，并预测到经典的RRACH(R=A/G, H=A、T、C) motif。m6A修饰相关的CSF3R和IFIT5在SLE中明显异常表达，其中IFIT5干扰后Jurkat细胞凋亡比例上升且细胞增殖能力下降。研究初步发现m6A修饰相关的mRNAs可能影响T细胞稳定性，但其中具体的调控机制仍需进一步研究。SLE疾病中存在多种目前已报道的m6A修饰酶，其中包括甲基转移酶、去甲基化酶和识别蛋白，因此m6A修饰可能动态调控SLE的疾病过程。甲基转移酶和去甲基化酶在SLE疾病中均呈现表达下调，其中甲基转移酶METTL3和去甲基化酶ALKBH5与SLE患者临床指标和治疗药物均存在关联。对METTL3基因干扰和过表达后发现，METTL3 表达下调会促进Jurkat细胞调亡，抑制Jurkat细胞增殖能力。ALKBH5过表达会促进T细胞凋亡，抑制T细胞凋亡。METTL3和ALKBH5以及其它差异表达的m6A甲基化酶在SLE疾病中的作用及其机制值得更深入探讨。