脊髓水平microRNA-219及其表观遗传修饰调控慢性炎性疼痛机制

The development of new effective therapy techniques and drugs for chronic pain depends on exploring the mechanisms of chroinc pain. Spinal neuronal sensitization contributed to the induction and maintenance of chronic pain. Neuronal sensitization 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. Serveral studies have shown that microRNA as a post-transcriptional regulator may be involved in many physiological and pathological processes including chronic pain through regulating the expression of related genes.Through using high-throughput sequencing method, we found that miR-219 in spinal cord was decreased significantly in a complete Freunds adjuvant-induced chronic inflammatory pain model. Furthermore, intrathecal injection of miR-219 mimics could markedly reverse chronic pain behavior. Bioinformatic analysis and luciferase assays confirmed that CaMKII gene is one of the target mRNA of miR-219. Approximately 15% of miR-219 gene promoter area is associated with CpG islands, which could be modified by epigenetic DNA methylation. In this proposal, we aim to investigate the roles and its mechanisms of spinal miR-219 and its epigenetic modification through behavioral, cellular and molecular studies in a complete Freund's adjuvant-induced chronic inflammatory pain model.

慢性疼痛仍缺乏有效治疗手段，理想治疗方法和药物的开发依赖于对慢性疼痛发生机制的认识。脊髓神经元可塑性改变被认为是慢性疼痛发生和维持的关键环节，神经元可塑性改变很大程度上依赖于神经元膜上受体、离子通道和胞内信号转导通路的表达改变，而这些改变又依赖于各自基因的表达调控，但慢性疼痛的基因调控机制仍不清楚。MiRNA是近年来发现的基因表达调控的一种方式，它参与调节多种生理和病理过程。最近我们利用高通量测序方法发现慢性疼痛状态下脊髓背角miR-219显著下调，且过表达miR-219可以逆转慢性痛行为，信息学分析发现与神经元可塑性和慢性疼痛密切相关的CaMKII可能是miR-219下游靶标，而miR-219启动子区存在可被甲基化表观遗传修饰的CpG岛。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miR-219及其表观遗传修饰对脊髓突触可塑性和痛行为的调节机制，为研发理想镇痛药物提供新的靶标。

慢性疼痛仍缺乏有效治疗手段，理想治疗方法和药物的开发依赖于对慢性疼痛发生机制的认识。脊髓神经元神经元膜上受体、离子通道和胞内信号转导通路的表达改变是慢性疼痛发生的关键环节，而这些改变又依赖于各自基因的表达调控，但慢性疼痛的基因调控机制仍不清楚。MiRNA是作为基因表达调控的一种方式，能够参与调节多种生理和病理过程。我们基于前期发现CFA诱导的慢性疼痛状态下脊髓背角miRNA-219显著下调，且过表达miRNA-219可以逆转慢性痛行为，信息学分析发现与神经元可塑性和慢性疼痛密切相关的CaMK2g可能是miRNA-219下游靶标，而miRNA-219启动子区存在可被甲基化表观遗传修饰的CpG岛。因此，本项目主要研究了以下内容：（1）脊髓神经元miRNA-219能调控慢性炎性疼痛；（2）miRNA-219能通过CaMK2g参与疼痛过程，且该过程脊髓神经元通过敏化介导疼痛发生；（3）疼痛过程中脊髓神经元 miRNA-219启动子区域会发生甲基化修饰，该表观遗传修饰能参与疼痛发生过程。本课题进行上述研究中，主要通过应用CFA诱导的慢性炎性疼痛小鼠为模型，通过逐步揭示脊髓神经元miRNA-219的甲基化修饰，降低miRNA-219其表达，继而导致其靶标CaMK2g表达增加，再通过上调脊髓神经元膜上NR1活性，最终导致神经元活化；动物行为学上则表现为痛觉敏化现象。总之，该研究从整体、细胞和分子水平阐明miRNA-219及其表观遗传修饰通过CamK2g对慢性炎性疼痛行为的调节机制。miRNA-219及其表观遗传修饰通过CamK2g对慢性炎性疼痛行为的调节机制发表于The Journal of Neuroscience (2014, 34(29): 9476–9483)。包括miRNA-219在内的miRNAs表观修饰作为疼痛治疗新靶点方面的研究，已获相关国家发明专利授权3项（授权号ZL201310247939.3；ZL201310279835.0；ZL200910032 410.3）。此外，深入探讨miRNA的去甲基化参与伤害性疼痛调节过程的研究结果发表于The Journal of Neuroscience (2016, 36(9): 2769–2781)。 因此，该课题阐明了 miRNA-219甲基化修饰参与疼痛的发生表观遗传机制，为研发理想镇痛药物提供新的靶点。