miR-181a-5p/YTHDF2/TRPV4信号通路在炎症性疼痛中的作用和机制研究

Osteoarthritis patients consider pain the most hampering symptom. However, the currently available treatments for OA related pain has limited effect even causes severe side effects. Previous studies have shown that miRNAs participated in the development of pain. Recent studies in our lab indicated that miR-181a-5p expression was up-regulated in the dorsal horn of CFA rats and inhibition of miR-181a-5p relieved mechanical hyperalgesia of CFA rats. Bioinformatics predicted an interaction between miR-181a-5p and the expression of m6A reader YTHDF2. We found that the expression of YTHDF2 was significantly decreased in the spinal dorsal horn of rats with CFA injection. The expression of TRPV4 was notably increased in the spinal cord dorsal horn of CFA rats. Several methylation sites for m6A have been found in TRPV4 pre-mRNA by bioinformatics prediction. Based on these results, we proposed the following hypothesis: in the CFA-induced osteoarthritis pain rats, miR-181a-5p was increased and inhibited the expression of YTHDF2, elevated TRPV4 expression, enhanced the function of TRPV4 and increased the neuronal excitability, leading to inflammatory pain hypersensitivity. The purpose of this study was to elucidate the role of miR-181a-5p /YTHDF2/TRPV4 signaling pathway in inflammatory pain. We hope that our study will further explain the pathological mechanisms of osteoarthritis pain and provide a new therapeutic target for clinical treatment for chronic inflammatory pain in osteoarthritis patients.

炎症性疼痛是骨关节炎患者的重要临床症状之一，发病机制尚未阐明，治疗手段短缺。已有研究表明miRNAs参与疼痛的发生发展过程。我们的前期研究发现在CFA大鼠炎症性疼痛模型中，miR-181a-5p在脊髓背角中的表达明显上升，抑制miR-181a-5p能够缓减大鼠痛觉过敏。生物信息预测发现miR-181a-5p与m6A甲基化识别酶YTHDF2相互作用。TRPV4在CFA大鼠脊髓中的表达明显增加，而TRPV4 pre-mRNA有多个m6A甲基化位点。由此提出以下假设：miR-181a-5p通过抑制YTHDF2，降低了YTHDF2对m6A+ TRPV4 mRNA的降解，提高了TRPV4的表达，增强了TRPV4的功能，增加了神经元的兴奋性，从而参与炎症性痛觉过敏。本课题旨在阐明miR-181a-5p/YTHDF2/TRPV4信号通路在炎症性疼痛中的作用机制，为临床治疗提供理论依据和新的治疗靶点。

骨关节炎是世界上最常见和致残的关节炎之一，引起关节疼痛，肿胀，僵硬及功能障碍。炎症性疼痛是骨关节炎患者的重要临床症状之一，是骨关节炎患者认为最难忍受的临床症状，其发病机制尚未阐明，治疗手段短缺。本研究旨在探讨炎症性疼痛大鼠脊髓背角miR-181a-5p/YTHDF2/TRPV4信号通路参与炎症性痛觉过敏的调控机制。本项目研究通过RT-PCR和western blot检测miR-181a-5p、YTHDF2和TRPV4在CFA大鼠脊髓中的表达，分析其与慢性炎症性疼痛的相关性。原位杂交检测miR-181a-5p的表达与定位，免疫荧光检测YTHDF2、TRPV4的表达与定位。通过鞘内注射miR-181a-5p antagomir/agomir或YTHDF2过表达/干扰慢病毒观察CFA大鼠疼痛反应及下游分子表达的影响。通过双荧光素酶报告基因检测miR-181a-5p和YTHDF2的直接相互作用。体外细胞实验检测调控miR-181a-5p对YTHDF2表达的影响，调控YTHDF2对TRPV4表达的影响。结果发现在CFA大鼠炎症性疼痛模型中，miR-181a-5p在脊髓背角中的表达明显上升，通过抑制YTHDF2，降低了YTHDF2对m6A+ TRPV4 mRNA的降解，提高了TRPV4的表达，增强了TRPV4的功能，增加了神经元的兴奋性，从而参与炎症性痛觉过敏，为临床治疗提供新的治疗靶点。