DNMT1介导的DNA启动子甲基化修饰调控DM难愈创面miRNA203表达和ESCs增殖、分化的作用机制

Preliminary studies from our group and others suggested that skin-specific miRNA203 was highly expressed and involved in the regulation of proliferation, differentiation and apoptosis of epidermal stem cells (ESCs) in impaired wound healing of diabetic mellitus (DM). Therefore, the abnormal expression of miRNA203 may be one of the important mechanisms of wound's poor healing. However, the mechanism of increased miRNA203 expression of refractory DM wound remains unclear. Further research reveals the fact that the expression of DNMT1(DNA methyltransferase 1) in DM refractory wounds is aberrant and the miRNA203 promoter remains hypomethylated. Based on the existing research, we hypothesize that hyperglycemia might inhibit miRNA203 expression through DNA promoter methylation via DNMT1 and then regulate the proliferation, differentiation of ESCs via downstream target genes and signal pathways , thereby inhibit wound healing. By using Chromatin immunoprecitation, lentivirus transfection and transgene technology both in vivo and vitro, we aim to investigate the epigenetic regulation of miRNA203, definitize the molecular mechanism of DNMT1/DNA promoter methylation/miRNA203 and Wnt/β-catenin signaling pathway regulating DM wound healing process. Through the perspective of epigenetics, this project will reveal novel molecular mechanisms underlying diabetic wound healing and provide new targets for designing effective therapeutic strategies, deepen the understanding of the mechanism of the formation of refractory DM wounds, and may provide new insights for its treatment.

前期我们首先报道皮肤特异的miRNA203在DM难愈创面高表达,且参与ESCs增殖和分化的调控。因此miRNA203的表达异常可能是DM创面难愈的重要原因。但其具体调控机制仍不清楚。进一步研究发现DM难愈创面中DNMT1表达异常,且miRNA203启动子区域呈低甲基化水平。因此，我们推测高糖环境可能通过DNMT1介导的DNA启动子甲基化修饰影响miRNA203的表达，继而激活下游的靶基因及潜在信号通路调控ESCs增殖分化、导致DM创面难愈。本项目拟采用ChIP、慢病毒双向调控及转基因小鼠等技术在体内、外逐级验证DNMT1/DNA启动子甲基化/miRNA203和下游Wnt/β-catenin信号网络影响DM创面愈合的作用机制。将从表观遗传学机制的角度阐明miRNA203表达的上游调控机制和下游的信号通路，并发现作用靶点的潜在治疗价值，从而为DM难愈合创面的治疗提供新的理论基础和临床策略。

研究背景：前期我们首先报道皮肤特异的miRNA203在DM难愈创面高表达,且参与ESCs增殖和分化的调控。因此miRNA203的表达异常可能是DM创面难愈的重要原因。但其具体调控机制仍不清楚。进一步研究发现DM难愈创面中DNMT1表达异常,且miRNA203启动子区域呈低甲基化水平。因此，我们推测高糖环境可能通过DNMT1介导的DNA启动子甲基化修饰影响miRNA203的表达，继而激活下游的靶基因及潜在信号通路调控ESCs增殖分化、导致DM创面难愈。.研究内容：单细胞转录组测序确定ESCs增殖分化障碍可能是DM创面难愈的重要因素；组织水平验证DM难愈创面中DNMT1表达水平下调并抑制ESCs的增殖分化能力；细胞水平验证DNMT1对ESCs的分子生物学功能；组织及细胞水平探索DNMT1对miR203表达的调节作用；DNMT1/miR203轴调控ESCs的增殖分化能力的下游机制探索；In Vivo探索DNMT1对db/db小鼠创面愈合的作用.重要结果：DM创面中的ESCs存在增殖分化障碍，且可能与BC-2和DAK有关；而这两种细胞上调与促炎相关的TF，下调与促增殖分化相关的TF，提示ESCs增殖分化障碍可能是DM创面难愈的重要因素。随后证实DM创面中的ESCs存在增殖分化障碍可能与下调的DNMT1相关。细胞实验证实DNMT1通过甲基化miR203基因启动子抑制其表达进而增强ESCs的增殖，迁移及干性维持能力。下游机制探索表明DNMT1/miR203轴可通过直接靶向PIK3CA进而影响ESCs内PI3K/AKT/mTOR信号通路的激活。动物实验表明DNMT1在体内通过维持ESCs的干性，加快新生肉芽的生长及表皮化速度进而促进DM创面的愈合。.关键数据及其科学意义：DNMT1/miR203/PIK3CA信号轴通过调节PI3K/AKT/mTOR信号通路影响ESCs的增殖分化能力从而影响创面愈合，我们的发现为DM难愈创面的治疗提供了新的治疗靶点。