miRNA-22及其m6A甲基化修饰通过正调控Mtf1介导慢性炎性疼痛的分子机制研究

Chronic pain still lacks specific effective drugs in treatment. Recent findings suggest that miRNA, a new regulatory mechanism of genes expression, plays an important role in the development and maintenance of chronic pain. In the preliminary experiment, we found that spinal miRNA-22 was significantly increased in complete Freund's adjuvant (CFA)-induced chronic inflammatory pain model in mice. Knockdown of miRNA-22 alleviated the thermal hyperalgesia and mechanical allodynia. Secondly, miRNA-22 was predictively bound to the promoter of Mtf1, and positively regulated the Mtf1 expression. Mtf1 was previously reported to be involved in the modulation of neuronal excitability via targeting Cav3.2 channel. Thirdly, CFA-induced chronic inflammatory pain increased the N6-methyladenosine (m6A) of pre-miRNA-22. Finally, knockdown of Mettl3 reversed the increased m6A level of pre-miRNA-22 and the expression of miRNA-22. Taken together, the purpose of this study is to investigate whether miRNA-22 and its m6A modification could regulate chronic inflammatory pain through positively targeting Mtf1 at the spinal cord level. This study will reveal a new functional regulatory epigenetic mechanism underlying chronic inflammatory pain processing, which will provide a possible target for the development of analgesic drugs.

慢性疼痛是一种疾病, 其发病机制复杂，临床上仍缺乏有效的治疗手段。近年发现miRNA参与慢性疼痛发展过程，但其确切机制仍不清楚。预实验表明，慢性疼痛状态下，脊髓背角miRNA-22表达显著增加，且下调miRNA-22能有效缓解疼痛症状；信息学分析及体外验证显示，miRNA- 22可能结合到Mtf1启动子正调控其表达；Mtf1则可能通过Cav3.2介导神经元兴奋性过程。进一步结果表明，疼痛下miRNA-22前体腺嘌呤甲基化（m6A）水平增加，下调m6A甲基化转移酶Mettl3能逆转miRNA-22前体m6A水平及miRNA-22的增加。因此，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脊髓miRNA-22及其m6A表观遗传修饰通过正调控Mtf1/Cav3.2对脊髓突触可塑性和痛行为的调节机制，以期为理想镇痛药物的研发提供新的靶点和理论依据。

慢性疼痛是一种疾病, 其发病机制复杂，临床上仍缺乏有效的治疗手段。近年发现包括miRNA在内的表观遗传修饰参与慢性疼痛发展过程，但其确切机制仍不清楚。RNA的m6A甲基化修饰丰富存在于中枢神经系统，是基因表达调控新方式。本研究应用CFA慢性炎性痛小鼠模型，围绕脊髓水平RNA的m6A甲基化修饰能否参与疼痛调控过程，从整体、细胞和分子水平研究、阐明了脊髓水平m6A甲基化修饰及其调控蛋白Mettl3参与慢性疼痛发生和维持的表观遗传修饰新机制。我们的研究取得以下三个成果：1）阐明了脊髓水平Mettl3通过调控DNA去甲基化酶Tet1参与慢性疼痛的分子机制；2）阐明了脊髓miRNA-22通过正调控Mtf1介导慢性炎性疼痛的分子机制；3） 揭示了miRNA-1224以Ago2依赖方式调控circRNA-Philip1l前体剪接介导参与慢性疼痛调控。该课题研究的完成不仅在学术上拓展了RNA的甲基化修饰神经生物学领域，而且为基于RNA的m6A甲基化修饰用于慢性疼痛临床治疗和药物研发提供新的靶标。上述研究成果分别在Journal of Neuroscience（被选为当期重点推荐文章）、Pain（当期配发长篇述评文章和视频介绍）和Oxidative Medicine and Cellular Longevity等SCI 论文；获得课题相关授权国家发明专利2项。