miR-503调控皮肤创伤修复增殖期向重塑期转换的作用与机制研究

It is gradually accepted that common cellular and molecular mechanisms are active in wounds and in cancer tissue. However, in contrast to cancer tissue, the process is "self-limiting" in healing wounds, resulting controlled cell proliferation, differentiation, migration and remodeling. While, this kind of "self-limiting", i.e. capability of transition from the proliferation phase to remodeling phase during wound healing, is still unclear in molecular level. Previously, miR-503, a microRNA with anti-proliferation and pro-apoptosis function, was found induced consistently with high expression level during the post-proliferation phase of cutaneous wound healing. And so we have proposed the hypothesis as follow: miR-503 is induced with space-time specific expression during cutaneous wound healing, which could participate in "arrest" of wound healing by regulating the transition from proliferation phase to remodeling phase. In this project, the role of miR-503 during cutaneous wound healing will be elucidated systematically in both in vivo mouse and in vitro cell models by experimental approachs such as ISH、qRT-PCR、microarray analysis and so on. The space-time expression and cellular localization of miR-503 in the granulation tissue, and its relation to pathological change of repairing skin, will be investigated firstly. Then, the effective intervention strategies of miR-503 exprssion in both in vivo and in vitro models will be established, and the impacts of both gain-of-function (GOF) and loss-of-function (LOF) on in vitro cell proliferation, migration, apoptosis and in vivo wound healing will be evaluated. At last, the functional target genes of miR-503 will be explored by combination microarray with reporter gene analysis. In brief, results of this project will bring something new in the theoretical field of wound healing, and what's more, it will provide the theoretical basis and new target for promoting wound healing in clinical practice.

创伤修复与肿瘤组织有相似细胞与分子机制，本质区别在于前者具有"自限性"。而这种"自限性"，即修复增殖期向重塑期转换的分子机制知之甚少有待探索。前期发现miR-503在皮肤修复增殖后期呈持续高表达，结合miR-503抑增殖、促凋亡报道，提出创伤修复中miR-503时空特异性高表达，参与修复"制动"，调控增殖期向重塑期转换的假说。项目拟采用在体小鼠皮肤创伤和体外细胞模型，选用原位杂交、定量PCR、芯片分析等方法，系统研究皮肤愈合中miR-503表达的时空特点、细胞定位及与皮肤修复病理的关系；在体和离体模型上，建立GOF/LOF策略干预miR-503水平，观察对修复细胞增殖、迁移、凋亡，和创面愈合影响；结合基因芯片和报告基因-候选UTR技术筛选miR-503靶基因，从而阐明miR-503参与皮肤创面愈合的作用与机制。研究结果可望深化对创伤愈合的理论认识，为临床干预损伤修复提供理论依据和新靶点。

前期比较创面修复增殖期肉芽组织与正常皮肤的miRNAs表达谱发现miR-503表达显著上调，结合其既往功能报道，推测其参与了创面修复增殖期向重塑期的转换，围绕着阐明miR-503及miR-205在创面修复中的作用与机制我们开展了系列研究。通过在体、离体的系列实验初步探索了miR-503及miR-205参与创面修复调控的作用。针对miR-530的主要结果发现为，（1）通过定量PCR明确miR-503有别于所在家族其他成员在修复增殖期表达上调最高，与其同簇的miR-322和miR-351也有不同程度的上调；原位杂交显示miR-503在肉芽的表皮和间质均有明显的上调，呈广泛性表达。（2）初步研究显示创面局部上调miR-503水平促进增殖期细胞的消退，而miR-503-322-351簇的KO小鼠增殖期细胞消退减缓，提示其是促进增殖期向重塑期转换的调控分子。（3）体外皮肤成纤维细胞研究表明，生长因子去除（血清饥饿）能够诱导miR-503显著上调表达，而miR-503过表达能抑制细胞的自噬，提示miR-503可能通过自噬调节参与创面修复。针对miR-205的主要结果发现为，（1）通过原位杂交确定miR-205在创面迁移上皮表达下调，明确这一特殊表达模式。（2）体外细胞和在体创面局部抑制miR-205水平显著促进细胞迁移和创面再上皮化。（3）鉴定出ITGA5是miR-205发挥作用的重要功能靶标。（4）以静脉性溃疡为代表的难愈性创面存在ITGA5和miR-205的表达失调，可能参与了难愈性的形成。本研究阐明了miR-205是皮肤创面愈合过程中再上皮化调节的重要分子，初步明确了miR-503是调控修复由增殖期向重塑期转换的重要分子，更为深入的分子机制研究和可能的应用价值有待进一步阐明。