miRNA203调控ESCs分化促进DM难愈创面愈合的分子机制

The skin-specific miRNA203 is involved in the regulation of epidermal stem cell (ESCs) proliferation and differentiation.ESCs is the key cell of wound repair. We previously found the expression of miRNA203 showed a marked upregulation in diabetes mellitus（DM）skin wounds in rats, and we proposed the hypothesis that the abnormal expression of miRNA203 result in the disorders of ESCs proliferation and differentiation and DM delayed wound healing. The study is to systematically investigate spatial and temporal changes of miRNA203 expression,epigenetic modification,the relationship of the related gene expression and pathways during the process of DM wound healing in vivo DM wounds and in vitro ESCs model;to predict and validate new miRNA203 targets and preliminarily analyze their function by combining oligo chip and Target Scan bioinformatics; to intervene miRNA203 expression and observe the effect of the overexpression and downregulation of miRNA203 to the proliferation,adhesion and migration of ESCs and the change of related gene-expression profiling based on the GOF/LOF strategies of transfection; to establish miRNA203 transgenic mice model by microinjection and to test the in vivo effect of miRNA203 to DM chronic wound healing.The current work is likely to lead to clarify the mechanism of that miRNA203 regulate DM chronic wound healing and provide the theoretical basis and new therapeutic targets for the treatment of DM chronic wound.

皮肤特异表达的miRNA203参与ESCs增殖分化的调控，而ESCs是创面修复的关键细胞。我们前期发现miRNA203在DM小鼠皮肤创面表达显著升高，提出miRNA203的表达异常是导致ESCs增殖分化功能失调、DM创面难愈的假说。本项目拟在体皮肤和离体模型系统研究DM创面愈合过程中miRNA203的时序表达变化、表达的表观遗传学调控和修复相关基因的相互关系；联合oligo芯片和Target Scan生物信息学预测和确证miRNA203的新靶基因，并初步分析其功能；GOF/LOF干预策略研究过表达或敲低miRNA203对ESCs增殖、黏附与迁移能力及相关基因表达谱的变化；受精卵显微注射法构建miRNA203表达功能型和抑制型的转基因小鼠,在体验证miRNA203对转基因DM小鼠创面愈合的影响,阐明miRNA203调控ESCs增殖分化和DM创面愈合的机制，为该病的治疗提供理论基础和新靶点。

项目背景：.糖尿病（diabetes mellitus，DM）慢性难愈创面是DM患者的严重并发症之一，发生率高，预后差，不仅给患者身心造成很大痛苦，也给家庭和社会带来沉重的负担。目前DM创面难愈的机制尚不清楚，临床也缺乏有效的治疗方法。皮肤发育和创面愈合的每个特定阶段都需要在时空上有精准的基因表达调控。包括本课题组在内的研究结果证实，一些miRNAs特别是miRNA203与表皮干细胞（epidermal stem cells，ESCs）的增殖、分化、功能和病理改变密切相关。而ESCs是创面修复的关键性功能细胞，参与愈合过程的每个阶段。因此，我们推测皮肤中特异的miRNA表达异常可能导致ESCs的增殖分化功能失调而在DM难愈创面的愈合中起重要作用。.主要研究内容：.在体研究皮肤组织miRNA203表达量与DM创面严重程度的相关性；研究DM大鼠创面愈合过程ESCs功能、miRNA203表达、愈合相关基因及通路表达的变化；离体研究高糖作用下ESCs功能、miRNA203表达、愈合相关基因及通路表达的变化；研究干预miRNA203对ESCs功能、愈合相关基因和通路表达的作用；基因表达谱技术分析miRNA203作用的新靶基因和潜在的信号通路；下调miRNA203对DM大鼠创面愈合的影响。.重要结果：.DM创面皮肤组织较正常皮肤组织高表达miRNA203，miRNA203表达水平与DM创面严重程度呈正相关。DM大鼠创面愈合过程ESCs数量减少，增殖能力减弱，Wnt、Notch信号通路表达下调，miRNA203高表达。高糖作用下ESCs活性降低，增殖能力减弱，miRNA203高表达，Wnt、Notch信号通路表达下调；抑制miRNA203表达，可改善高糖的这一影响。ROCK2、MAPK8、MAPK9、PRKCA可能是miRNA203的新靶基因。通过拮抗在DM大鼠创面皮肤组织高表达miRNA203，可促进DM大鼠创面愈合，其机理可能是改善了ESCs功能，上调了Wnt、Notch信号通路表达。.关键数据及其科学意义：.通过拮抗在DM大鼠创面皮肤组织高表达miRNA203，可促进DM大鼠创面愈合，其机理可能是改善了ESCs功能，上调了Wnt、Notch信号通路表达。进而说明miRNA203在DM难愈创面的发病机制中发挥了重要作用。