miRNA203-Jagged1-Hes1信号轴调控ESCs在瘢痕形成中的作用机制研究

The skin-specific miRNA203 regulates the proliferation and differentiation of epidermal stem cells (ESCs), which are critical for the formation of hypertrophic scar. However, the mechanism of miRNA203 in regulating hypertrophic scar formation is not clear. We previously found the miRNA203 can activate Jagged1 signaling and elevate Hes1 expression during the formation of hypertrophic scar. Moreover, Jagged1 and Hes1 are targets of miRNA203 indicated by Target Scan and Pathway bioinformatics prediction. . Thus, we proposed that the abnormal expression of miRNA203 affects Hes1 expression and Jagged1 signaling pathway, resulting in proliferation and differentiation disorders of ESCs during the formation of hypertrophic scar in wound healing. In this study, we will take some molecular biology techniques such as RT-PCR, western blotting, siRNA, immunofluorescence, GOF (gain-of-function)/LOF (loss-of-function) strategies of transfection, and transgenic mice model by microinjection to acquire evidence of miRNA203-Jagged1-Hes1 on the regulation of epidermal stem cells (ESCs) proliferation and differentiation during the formation of hypertrophic scar in vivo and vitro. The work is likely to lead to clarify the mechanism of miRNA203-Jagged1-Hes1 on ESCs proliferation and differentiation and the formation of hypertrophic scar and provide the theoretical basis and new therapeutic targets for the treatment of hypertrophic scar.

ESCs的增殖和分化与瘢痕形成密切相关。文献和我们既往研究证实皮肤特异的miRNA203表达异常是导致ESCs增殖分化紊乱、瘢痕形成的重要原因，但调控机制不清楚。前期工作发现瘢痕形成中miRNA203可以激活Jagged1通路和下游基因Hes1的表达；采用靶基因预测和Pathway分析软件发现miRNA203的靶基因可能是Hes1，而Jagged1信号是其发挥作用的特异性通路；进一步生物信息学证实Hes1基因3’UTR存在miRNA203结合位点。因此，我们推测：创面愈合过程中miRNA203通过激活Jagged1信号通路并靶向下游Hes1基因调控ESCs增殖、分化，最终导致瘢痕形成。本课题将采用siRNA、GOF和LOF、蛋白印迹、质粒转染及构建转基因小鼠等技术，系统研究miRNA203-Jagged1-Hes1信号轴在瘢痕形成中的作用机制，为临床治疗瘢痕提供理论依据和新靶点。

项目背景：.ESCs的增殖和分化与瘢痕形成密切相关。文献和我们既往研究证实皮肤特异的miRNA203表达异常是导致ESCs增殖分化紊乱、瘢痕形成的重要原因，但调控机制不清楚。前期工作发现瘢痕形成中miRNA203可以激活Jagged1通路和下游基因Hes1的表达；采用靶基因预测和Pathway分析软件发现miRNA203的靶基因可能是Hes1，而Jagged1信号是其发挥作用的特异性通路；进一步生物信息学证实Hes1基因3’UTR存在miRNA203结合位点。因此，我们推测：创面愈合过程中miRNA203通过激活Jagged1信号通路并靶向下游Hes1基因调控ESCs增殖、分化，最终导致瘢痕形成。.主要研究内容：.检测正常皮肤和瘢痕皮肤中miRNA203表达变化和分布特点；研究miR-203与Hes1基因的3’-UTR相互作用调节Hes1的表达情况；分析ESC及MFB特异性标记物，研究MiR-203在促进ESCs向MFB细胞的分化的过程；体外研究miR-203对Notch1/Jagged1-Hes1信号通路及MFB的影响；研究Anti-miR-203治疗方法对伤口愈合及体内疤痕的形成的影响；研究Anti-miR-203治疗促进再上皮化、皮肤附着再生和胶原再分配的作用；研究Anti-miR-203治疗抑制体内ESC向MFB分化的背后的机制。.重要结果：.瘢痕皮肤中miRNA203的表达丰度是正常皮肤组织的8-10倍；miR-203下调野生型Hes1 mRNA的3’-UTR报道基因；合成性前体miR-203处理后显著降低了ESC表面标记物的表达，显著增加了MFB表面标记物表达；抗miR-203治疗后结果相反；用LV-Hes1预诱导ESC可以逆转合成性前体miR-203的影响； miR-203过表达可延迟伤口愈合，促进瘢痕形成；抗miR-203治疗后提高了创面愈合质量；抗miR-203治疗显著增加Hes1表达，降低α-SMA表达，其机理可能是miR-203通过降低Hes1的表达来促进ESC向MFB的分化。.关键数据及其科学意义：.通过拮抗在小鼠创面皮肤组织高表达miRNA203，可促进小鼠创面愈合，改善愈合质量