Mettl3介导lncRNA DGCR5 m6A甲基化调控miR-320靶向FOXM1在急性脊髓损伤中的机制研究

Acute spinal cord injury ( ASCI ) is a common disorder and has a high risk of disability. The non-coding RNA regulation of apoptosis-related genes plays an important role in the process of apoptosis of neurons in ASCI. Our previous study has revealed that lncRNA DGCR5 was significantly downregulated in ASCI neurons and interaction of lncRNA DGCR5 and PRDM5 could regulate neuron apoptosis to improve the ASCI, but the regulation mechanism of its expression and whether the regulation is modulated via ceRNA mechanism remains unclear. Based on bioinformatics methods, we have identified that lncRNA DGCR5 possesses m6A methylation site and could competing interact with miR-320 to regulate the expression of FOXM1. This project intends to use the techniques of reporting gene analysis, RNA pull down, RIP and so on to reveal the regulatory mechanism of Mettl3-mediated m6A methylation modification to lncRNA DGCR5 expression, and to elucidate lncRNA DGCR5/miR-320/FOXM1 axis regulates the molecular mechanism of neuron apoptosis, and demonstrates the role of the regulatory axis in ASCI repair in animal models. This study provides a new idea and a new target for the study of the regulation mechanism of ASCI repair.

急性脊髓损伤（ASCI）是一种常见且致残率高的疾病，非编码RNA调节凋亡相关基因在ASCI神经元凋亡过程中发挥重要作用。我们前期研究证明lncRNA DGCR5在ASCI神经元表达下降，能够与PRDM5互作调控神经元凋亡改善ASCI的损伤程度，但lncRNA DGCR5的表达调控机制及其能否通过ceRNA模式发挥调控作用还不清楚。生物信息学分析发现，lncRNA DGCR5序列上含有m6A甲基化位点，且可能通过竞争性结合miR-320靶向调控FOXM1。本项目拟运用报告基因分析、RNA pull down、RIP等技术，揭示Mettl3介导的m6A甲基化修饰对lncRNA DGCR5表达的调控机制，阐明lncRNA DGCR5/miR-320/FOXM1 轴调控神经细胞凋亡的分子机制，并于动物模型中论证该调控轴在ASCI修复中的作用。本研究为ASCI修复的调控机制研究提供了新思路和新靶标。

急性脊髓损伤（SCI）是一类严重的高致瘫性的中枢神经创伤，是神经创伤研究领域热点及难点，创伤后伤段脊髓局部神经元凋亡、神经轴突断裂和微循环障碍是 SCI 损伤加重，神经功能丧失的重要因素。我们前期研究证明lncRNA DGCR5在ASCI神经元表达下降，能够与PRDM5互作调控神经元凋亡改善ASCI的损伤程度，又通过生物信息学分析发现，lncRNA DGCR5序列上含有m6A甲基化位点，且可能通过竞争性结合miR-320靶向调控FOXM1。我们从动物实验和细胞学实验两个层面系统的研究了Mettl3介导的LncRNA DGCR5 m6A甲基化可通过改善 SCI 后局部微循环和抑制神经元凋亡等多方面促进脊髓损伤后神经功能的恢复及相关机制，揭示了LncRNA DGCR5-miR320-FOXM1通路在SCI后的作用机制：SCI后Mettl3的表达含量降低，导致LncRNA DGCR5的m6a甲基化下调，与mir320的结合降低，由于其与FOXM1基因竞争性结合在mir320上，所以FOXM1同mir320结合增加，导致游离的FOXM1基因减少，FOXM1蛋白的表达减少，所以FOXM1抑制神经细胞凋亡能力下降，导致脊髓损伤后的神经细胞凋亡增加，这一通路机制的研究为 Mettl3介导的LncRNA DGCR5 m6A甲基化 应用于 SCI 及相关脊髓疾病治疗的临床前疗效评估提供了实验科学依据。