长链非编码RNA H19调控IGF促进皮肤创伤愈合的作用及分子机制

Reparation of injured dermis is finely modulated by complicated network of gene expression, the mechanism of which is yet to be elucidated. Literatures published lately suggested that long noncoding RNA H19 (lncRNA H19) should take part in the expression-regulated procedure of insulin-like growth factors (IGFs). Also, reportedly, lncRNA H19 potentially promotes many kinds of cells to proliferate, migrate and differentiate. The analysis of structure bioinformatics shows that four conserved k-turn motifs lie in the 3'-region of lncRNA H19, which can bind with various protein, such as transcription factor Smads and mRNA-binding protein (IMP) et al. These molecules of protein play an important role to modulate the expression level of IGFs, at the transcription or translation level. Recently, a fact that the H19 gene is expressed at high level in accordance with IGFs in the regeneration tissue of surgical incision of mouse skin was disclosed by micro-array analysis in our previous research. Taken together, we postulate a hypothesis that a high level lncRNA H19, transcript-induced by trauma stress, up regulates the expression of IGFs through recruiting Smads et al, and then facilitates to wound healing of skin. So, this project will choice surgical incision skin of mouse and HaCaT cell as models to explore the temporal expression characteristics of H19 in the wound healing procedure, and its relation to skin regeneration by applying Northern blot, ChIP method, and so on. Further, the interaction of H19 RNA with Smads and IMP will be measured in vitro. Meanwhile, the influence of lncRNA H19 to IGFs' expression and epidermis phenotype cell will be investigated. Also, the function of facilitating wound healing of H19 will be tested though in vivo experiment, by over-expression or knock-down interference in the topical wound area of skin. Hopefully, the bio-function and mechanism of lncRNA H19 in wound healing of skin would be revealed, which may beneficial to explore better clinical measure for treatment of dys-healing of skin trauma.

皮肤损伤修复受复杂的基因表达网络调控，机制尚未阐明。新近研究提示，长链非编码RNA H19参与胰岛素样生长因子(IGFs)的表达调节和具有促细胞增殖、迁移等作用。基于H19 RNA具有可结合IGFs转录因子等蛋白分子的基序和我们芯片检测发现H19与IGFs在小鼠皮肤切割伤修复组织中一致性高表达的现象，提出创伤应激诱发高转录的H19 RNA募集Smads等分子上调IGFs的表达，从而促进创面愈合的假说。本项目拟采用小鼠切割伤皮肤和HaCaT细胞为模型，选用Northern blot、ChIP等方法，研究H19在皮肤创伤愈合中的时空表达特点及其与皮肤再生的关系；离体研究H19 RNA与Smads和mRNA转运蛋白IMP的相互作用及其对IGFs表达和细胞表型的影响；创面表达干预，在体验证H19的促愈作用。期望揭示H19在皮肤创伤愈合中的作用及分子机制，为难愈性创面的促愈干预提供理论依据和新靶点。

皮肤损伤修复是基因表达调控网络作用的复杂过程，其基因表达调控的分子机制尚远未阐明。本项目完成了以下研究内容：(1)长链非编码RNA H19（lncRNA H19）在皮肤和肌肉中的表达特点及其与创面愈合的关联性分析; (2) miR-205促进皮肤创面愈合的作用与分子机制；(3) lncRNA H19及其包含miR-675促进骨骼肌损伤修复的效果及机制。.表达分析发现小鼠皮肤创伤第3天创缘组织中lncRNA H19即显著上调，第7天达最高峰，第10天表达下降的特点。免疫组化揭示伤后lncRNA H19表达的动态变化与创面愈合间存在时间和空间的相关性。提示lncRNA H19参与皮肤创伤愈合过程。.miR-205在创伤皮肤生长边沿表达显著下调。细胞划痕实验表明抑制miR-205表达有促愈作用。增强或抑制miR-205实验证明miR-205调控皮肤创面愈合。表达实验表明miR-205负调控ITGA5基因。在体实验显示ITGA5在伤后第4天的创缘组织中表达明显提高，提示ITGA5参与创伤愈合过程。RNAi实验显示ITGA5表达抑制可抑制细胞划痕创面闭合，还可抵消抑制miR-205产生的促细胞移行效果。揭示miR-205通过负调控ITGA5的表达发挥促愈作用的机制。注射miR-205抑制分子能使创面提前2.5 d愈合。提示miR-205是潜在治疗难愈性创面的新靶点。.发现lncRNA H19及其包含的miR-675在肌肉中高表达。细胞实验显示miR-675表达在成肌分化前4天逐步升高，第5天开始下调，提示miR-675调控骨骼肌细胞分化。损伤股四头肌中lncRNA H19和miR-675的表达均在伤后前3天逐渐降低，然后逐渐升高（第7d升高最为明显）。RNAi实验显示lncRNA H19表达降低时，miR-675的表达也明显降低。敲低lncRNA H19的表达时，Myog和MHC的转录也显著降低；miR-675的表达增强后，Myog和MHC的转录和翻译表达即可恢复。揭示了miR-675通过Myog和MHC促进成肌分化。细胞实验表明miR-675-3p和miR-675-5p分别负调控Smad1、Smad5和Cdc6的转录和翻译。明确了miR-675通过负调控Smad1、Smad5和Cdc6的机制促进骨骼肌损伤修复的作用。提示miR-675是治疗肌肉损伤的新靶标。