Mettl3介导m6A甲基化RNA调控慢性炎性疼痛机制研究

Chronic pain is a frequently-occurring and refractory disease.The pathogenesis of chronic pain is complex, resulting in a lack of specific effective techniques and drugs in treatment. The lasting central sensitivity is a major cause in the development and maintenance of chronic pain, the plasticity of spinal cord is a core in the induction and maintenance of central sensitivity. Spinal plasticity mainly depends on the abnormal integration of membrane receptors, ion channels and intracellular signal transduction pathway mediated by modification of pain-related genes expression. However, the mechanisms of gene modification underlying chronic pain still keep unclear. Several studies have shown that adenosine methylation in RNA (m6A) mediated by Mettl3 may be involved in the process of physiology and pathology. Spinal Mettl3 was increased in complete Freunds adjuvant-induced chronic inflammatory pain mice, and downregulation of Tet1 and Tet3 could reverse pain behavior. Furthermore, through using RNA immunoprecipitation and high-throughput sequencing method, we found that m6A level in the terminated region of DRD-mRNA was significantly increased in spinal cord of CFA-induced chronic pain mice. Therefore, DRD3 is a possible downstream gene regulated by Mettl3. In addition, DRD3 may change the neuronal synapse by negatively regulating Cavα2δ1 in the spinal cord of CFA-induced pain mice. In this proposal, we aim to investigate the roles and its mechanisms of spinal Mettl3 regulating chronic pain by targeting DRD3 through behavioral, cellular and molecular studies in a complete Freunds adjuvant-induced chronic inflammatory pain model.

慢性疼痛是多种原因诱发的高发性和顽固性病症，其发病机制复杂并缺乏特异性治疗措施。脊髓神经元可塑性改变则是慢性疼痛发生和维持的关键环节，而脊髓神经元可塑性改变则依赖于神经元膜上受体、离子通道和胞内信号转导通路异常变化，这些变化又依赖于各自基因表达调控，但慢性疼痛基因调控机制仍不清楚。Mettl3介导RNA腺嘌呤甲基化（m6A）是转录后基因表达调控新方式。最近，我们发现慢性炎性痛下，Mettl3表达增加，且下调Mettl3可以逆转慢性痛行为；脊髓背角多巴胺受体DRD3的mRNA终止密码子区域m6A增加显著，而DRD3蛋白表达降低，可能是Mettl3下游基因；DRD3则可能通过负调控Cavα2δ1介导脊髓神经元可塑性改变。本项目拟以慢性炎性痛为模型，从整体、细胞和分子水平阐明Mettl3通过DRD3对脊髓神经元突触可塑性和痛行为的调节机制，以期为慢性疼痛治疗提供新的药物靶点和理论依据。

（1）研究背景：近来研究表明，表观遗传学修饰与疼痛发生关系密切。RNA/DNA（去）甲基化作为基因表达调控的一种重要表观遗传学修饰方式，在神经疾病调控中发挥重要作用。然而，DNA羟甲基化及RNA甲基化修饰是否与疼痛有关联，尚不清楚。 （2）主要研究内容：本研究围绕TET介导的羟甲基化参与伤害性疼痛的调控为主线；深入讨论了基因羟甲基化通过调节miRNA-365（非编码RNA）及Stat3（DNA羟甲基化）的表达来参与疼痛调控表观遗传学机制。 （3） 重要结果：阐明了 (a) 脊髓水平TET通过miRNA-365或Stat3介导的疼痛调节及其机制；(b) 脊髓水平miRNA-1224通过Ago2依赖的方式调控circRNA-Philip1l的生成参与慢性炎性疼痛调控机制。（4）关键数据及其科学意义：TET1和TET3能介导Stat3或miRNA-365基因的羟甲基化修饰参与疼痛行为的调节机制，发表于Anesthesiology (2014, 34(29): 9476–9483)和The Journal of Neuroscience (2016, 36(9): 2769–2781)。并获相关国家发明专利授权2项（授权号 ZL201310247939.3；201310247940.6）。此外，深入探讨亦对其它非编码circRNA-Philip1l及其生成，参与慢性炎性疼痛调控机制；该结果已于2018年12月26日被Journal of Neuroscience接收。 总之，该项目阐明了RNA/DNA（去）甲基化修饰参与疼痛的发生表观遗传机制，这为研发理想镇痛药物提供新的靶标。此外，该研究还首次将RNA的（去）甲基化修饰引入到疼痛研究领域。