miRNA-9及其羟甲基化修饰通过Kcnc1调控慢性炎性疼痛的机制研究

Chronic pain is a frequently-occurring and refractory disease, and lacks specific effective drugs in treatment. However, the mechanism underlying chronic pain remains unclear. Therefore, the unraveling of DNA cytosine hydroxymethylation (5hmC) mediated by TET enzyme is a new regulatory mechanism in gene expression. In the preliminary experiment, we found: 1) Tet1 and Tet3 were increased in spinal cord of complete Freund adjuvant-induced chronic inflammatory pain mice, and knockdown of Tet1 and Tet3 alleviated the pain behavior; 2) chronic inflammatory pain induced the increase of spinal 5hmC in miRNA-9 promoter, which was reversed by the downregulation of Tet1 and Tet3; 3) Informatics analysis and in vitro verification showed that Kcnc1 is a predictive target gene of miRNA-9. Collectively, in this study, we aim to investigate whether miRNA-9 and its hydroxymethylation regulate chronic inflammatory pain via targeting Kcnc1. This study will reveal a new functional regulatory epigenetic mechanism underlying chronic inflammatory pain process, which will provide a possible target for the development of analgesic drugs.

慢性疼痛是一顽固高发性疾病，其调控机制仍不清楚。羟甲基化酶Tet介导基因羟甲基化修饰是基因表达调控的新方式。预试验表明慢性炎性疼痛下: 1）小鼠脊髓Tet1和Tet3 表达增加，下调Tet1和Tet3能有效缓解疼痛症状；2）高通量测序显示，miRNA-9启动子羟甲基化增加显著，且下调Tet1和Tet3能降低miRNA-9羟甲基化及其表达水平；3）信息学分析及体外验证显示，miRNA-9可能结合到Kcnc1钾离子通道mRNA的3’-UTR非翻译区，下调或上调miRNA-9则能增加或降低Kcnc1荧光素酶报告基因活性。综上，本项目拟以慢性炎性疼痛小鼠为模型，从整体、细胞和分子水平阐明miRNA-9及其羟甲基化修饰通过Kcnc1调控慢性炎性疼痛的表观遗传学机制，以期为理想镇痛药物研发提供新靶点和理论依据。

慢性疼痛由于缺乏临床有效治疗，严重影响了人们的生活质量，机制至今不是很明确。越来越多的证据表明，miRNAs通过切割或降解细胞浆中的靶mRNA参与疼痛过程。然而，神经元核miRNAs在慢性疼痛条件下是否以及如何参与基因调控仍是未知的。我们检测了完全弗氏佐剂(CFA)诱导的慢性炎症性疼痛小鼠脊髓中miRNA9和Kcnc1的表达水平，同时我们也检测了miRNA-22和Mtf1的表达水平。采用免疫荧光、ChIP、RT-qPCR、免疫印迹、行为检测等方法分别研究了miRNA9和miRNA-22对慢性炎性痛的调控机制。我们发现（1）Tet1 和 Tet3 丰富表达于脊髓神经元细胞，且慢性炎性疼痛下两 者表达增加（2）慢性炎性疼痛下脊髓背角 5hmC 变化显著（3）疼痛下脊髓 Tet1 和 Tet3 表达增加且能参与痛觉调制过程（4）CFA 炎性疼痛增加了 miRNA-9 启动子 5hmC 及 miRNA-9表达水平（5）下调 miRNA-9 表达减轻 CFA 炎性疼痛症状（6）Kcnc1 可能是 miRNA-9 调控靶标（7）疼痛下脊髓过表达Kcnc1能减轻小鼠疼痛症状。同时我们发现炎性疼痛小鼠脊髓神经元核中miRNA-22显著增加。敲除或阻断miRNA-22减轻CFA机械性触痛和热痛觉过敏;相反，过度表达miRNA-22模仿疼痛样行为。我们首先揭示了miRNA在转录水平上通过介导靶标特异性基因表达参与慢性疼痛，并提出了一种新的治疗慢性疼痛的策略