m⁶A甲基化修饰介导pri-miR-129识别加工在糖尿病皮肤伤口愈合中的作用及机制研究

miRNAs play an important role in the diabetic wound healing. Previous studies have confirmed that decreased expression of miR-129 family was an essential event contributing to refractory diabetic wound, which resulted from the inhibition of pri-miR-129 processing. It has been reported that N6-methyladenosine (m⁶A) mediated by METTL3 facilitates the recognition and processing of pri-miRNA by microprocessors (Drosha and DGCR8). But the reader of m⁶A in this procedure is still unknown. We speculate that low expression of METTL3 and YTHDC1 in keratinocytes may lead to decrease of m⁶A marks in pri-miR-129 and interrupt the "bridge" connecting with the microprocessor, which results in a significant decrease of the expression of mature miR-129 family. This project intends to discover evidence that m⁶A contributes to the low expression of miR-129 family in diabetic skin by MeRIP, fCLIP, RNA pull-down and other methods. Besides, it aims to reveal a new mechanism of decreased expression of miR-129 family in diabetic skin in order to provide scientific evidence for finding potential new targets for the treatment of diabetic woud.

miRNAs在糖尿病创面难愈中发挥重要作用。前期实验证实，miR-129家族低表达是影响糖尿病创面愈合的重要原因，并与pri-miR-129识别加工过程相关。文献报道，METTL3介导的m⁶A甲基化修饰有助于pri-miRNA被微处理器（Drosha与DGCR8蛋白组成）识别加工，但m⁶A如何被微处理器识别尚不明确。预实验表明可能与阅读蛋白YTHDC1相关。因此，我们提出：糖尿病状态下，角质形成细胞METTL3、YTHDC1低表达，可能导致pri-miR-129 m⁶A修饰减少，并中断与微处理器的连接“桥梁”，引起miR-129家族成熟体表达降低。本项目拟通过MeRIP、fCLIP、RNA pull-down等方法，获得m⁶A修饰减少导致糖尿病皮肤miR-129家族低表达的可靠证据，有望揭示糖尿病皮肤角质形成细胞miR-129家族低表达的新机制，为寻找糖尿病皮肤创面治疗新靶点提供实验依据。

前期实验证实，miR-129家族低表达是影响糖尿病创面愈合的重要原因，并与pri-miR-129识别加工过程相关。文献报道，METTL3介导的m⁶A甲基化修饰有助于pri-miRNA被微处理器（Drosha与DGCR8蛋白组成）识别加工，但m⁶A如何被微处理器识别尚不明确。预实验表明可能与阅读蛋白YTHDC1相关。我们研究发现，糖尿病状态下，角质形成细胞METTL3低表达，可能导致pri-miR-129 m⁶A修饰减少，并中断与微处理器的连接“桥梁”，引起miR-129家族成熟体表达降低。另外，我们研究发现m6A阅读蛋白YTHDC1在糖尿病皮肤低表达，其可以与自噬关键蛋白SQSTM1 的mRNA结合，维持角质形成细胞的修复功能，揭示了YTHDC1通过调节糖尿病角质形成细胞中SQSTM1核mRNA的稳定性来调节自噬的新功能。总之，本项目为寻找糖尿病皮肤创面治疗新靶点提供了实验依据。