RNA甲基转移酶METTL3介导的SOCS　m6A修饰在Graves病发病中的作用及机制

Graves' disease (GD) is a common autoimmune disease. Immune dysfunction caused by imbalance of immune cells plays a key role in the pathogenesis of GD. It is well-known that the epigenetic modifications in RNA exert critical roles in regulating the differentiation of cells through changing the expressions of some key genes, like SOCS family members and et al. Our preliminary research found that the key enzyme of mRNA N6-methyladenosine (m6A) methylation, METTL3, is aberrantly down-expressed in GD. However, it is still unclear whether METTL3 is involved in the development of GD through regulating the balance of immune cells. Hereby we hypothesize that METTL3 is intensively involved in the pathogenesis of GD, that the down-expressed METTL3 could mediate the mRNA m6A methylation- modification of SOCS family, then further induce the imbalance of key immune cells and finally results in GD. To validate this hypothesis, we will perform clinical research, in-vitro cell culture experiments and in-vivo animal experiments with GD animal models by using a variety of techniques, such as m6A-seq, CRISPR/Cas9 method, RNA interference and so on, to study the influence of METTL3 on GD through inducing the mRNA m6A modification of SOCS family members, and its potential molecular mechanisms, which will hopefully provide a novel target for the therapy of GD.

Graves病(Graves' disease, GD)是一种常见的自身免疫病。免疫细胞失衡触发的免疫紊乱在其发病中起关键作用。已知RNA表观遗传修饰通过影响SOCS等关键基因的表达，在细胞分化中发挥调控作用。我们前期研究也发现，介导RNA m6A甲基化修饰的关键酶METTL3在GD中表达降低，但是METTL3是否通过调控免疫细胞平衡参与GD的发生尚不明确。为此，我们提出假说：METTL3与GD的发生有关，其表达降低后可以通过介导SOCS家族mRNA m6A的甲基化修饰，从而引起免疫细胞失衡，最终触发GD。为了验证这一假说，我们拟采用临床研究、体外细胞培养和动物GD疾病模型的构建，综合运用m6A-seq、CRISPR/Cas9技术、RNA干扰等实验方法来研究METTL3介导SOCS mRNA m6A修饰对GD发生的影响及可能的分子机制，为GD提供潜在治疗靶点。

近年研究认为，免疫细胞失衡触发的免疫紊乱在Graves病（Graves' disease, GD）发病中起关键作用。已知RNA表观遗传修饰通过影响SOCS等关键基因的表达，在细胞分化中发挥调控作用。我们前期研究也发现，介导RNA m6A甲基化修饰的关键酶METTL3在GD中表达降低，但是METTL3是否通过影响SOCS mRNA的m6A甲基化修饰，从而参与GD的发生尚不明确。为此，我们采用临床样本研究和体外细胞培养方法，综合运用生物信息学和分子生物学等方法和技术来研究METTL3介导相关分子mRNA m6A修饰对GD发生的影响及可能的机制，为GD提供潜在治疗靶点。. 研究结果发现：.1、METTL3和SOCS家族分子在GD甲状腺组织中存在异常表达：GD甲状腺组织中METTL3 mRNA表达水平明显降低，而SOCS3 mRNA表达水平明显升高。.2、METTL3和其他多个mRNA m6A 甲基化修饰关键酶基因在GD患者CD4+T细胞中存在异常表达：GD患者CD4+T细胞中METTL3基因表达水平较健康对照者降低，其他如FTO、ALKBH5的表达水平也呈现出同样的下降趋势。.3、搜索相关数据库，结果发现：SOCS家族分子mRNA上存在数量较多的m6A 甲基化修饰位点，而m6A甲基化修饰关键酶可以通过介导m6A甲基化修饰调控SOCS家族分子的mRNA或蛋白表达水平。.4、临床样本研究m6A关键酶基因mRNA在GD外周血单个核细胞（PBMC）中的表达情况：METTL3 mRNA在GD患者PBMC中表达显著降低，而METTL14、FTO和 ALKBH5 mRNA表达水平无明显变化。.5、体外METTL3细胞敲除实验结果显示，SOCS家族分子SOCS1, SOCS2, SOCS4, SOCS5和SOCS6在METTL3敲除后其表达明显升高。.6、m6A甲基化关键酶基因METTL3存在多态性位点，且与GD易感性存在相关性。.7、m6A甲基化关键酶基因ALKBH5存在多态性位点，且与GD易感性存在相关性。. 我们将以上结果总结为：METTL3与GD的发生有关，其表达降低后可以通过介导SOCS家族mRNA m6A的甲基化修饰，从而参与GD的发病过程；另外，METTL3、ALKBH5存在基因多态性，且与GD发病风险相关。