先天性巨结肠发生新机制：miR-218-1介导肠神经嵴细胞迁移和增殖障碍

The pathogenesis of Hirschsprung's disease (HSCR) is highly associated with the migration and proliferation failure of enteric neural crest-derived cells (ENCCs), but the concrete mechanism is still unclear. MiR-218-1 was one aberrant up-regulated miRNA we found after screening the pathologic segment of HSCR patients by miRNA microarray. The bioinformatics analysis indicated that RET was the potential target gene of miR-218-1 while SLIT2 was its host gene. It was confirmed that miR-218-1 and SLIT2 were overexpressed and that RET was underexpressed in pathologic tissues of a large number of HSCR patients. It was found in vitro assay that the overexpression of miR-218-1 could suppress the expression of RET gene and restrict the migration and proliferation of nerve cells. Based on the findings above, we made the hypothesis that the consistently high expression level of miR-218-1 and its host gene SLIT2 would suppress the expression of RET gene, cause the migration and proliferation failure of ENCCs, and finally result in HSCR. Our research aims to investigate the mechanism of how miR-218-1 mediate RET expression and cause the disorder of migration and proliferation of ENCCs. Besides, we will explore whether miR-218-1 and its host gene SLIT2 suppress the migration and proliferation of ENCCs. Also, we will identify the biological function of miR-218-1 in the enteric nervous system formation process in vivo assay by the miR-218-1 transgenic mice. This research will give a new pathogenesis of HSCR, providing a potential target of prevention and treatment for HSCR in embryonic period.

肠神经嵴细胞（ENCCs）迁移和增殖障碍是先天性巨结肠（HSCR）发生的主要原因，但其具体机制尚不明确。我们用miRNA芯片筛选HSCR病变段时发现miR-218-1显著高表达。生物信息学显示RET是miR-218-1的靶基因，SLIT2是其宿主基因。人群验证发现miR-218-1、SLIT2在HSCR病变段高表达，RET低表达。体外实验提示miR-218-1抑制RET表达并影响神经细胞迁移增殖功能。由此设想：miR-218-1与其上游宿主基因一致性高表达，抑制靶基因RET表达，影响ENCCs向消化道迁移和增殖，引起HSCR。 本研究将明确miR-218-1介导RET影响ENCCs迁移增殖障碍中的作用；阐述miR-218-1与其宿主基因协同影响ENCCs迁移增殖；通过miR-218-1转基因小鼠，鉴定其在HSCR发生中的作用。本研究可为HSCR发生机制提供新视点和胚胎期防治提供潜在靶标。

肠神经嵴细胞（ENCCs）迁移和增殖障碍是先天性巨结肠（HSCR）发生的主要原因，但其具体机制尚不明确，本项目主要研究miRNA-218-1及其上下游通路在先天性巨结肠发生中的作用及机制。. 首先应用miRNA芯片筛选HSCR病变段肠管差异表达miRNA时发现miR-218-1的表达显著增高。生物信息学分析及双荧光素酶报告基因检测显示RET及PLAG1基因是miR-218-1的靶基因，SLIT2是其宿主基因。先天性巨结肠标本检测发现miR-218-1、SLIT2在HSCR病变段高表达，RET及PLAG1表达降低。体外细胞实验显示miR-218-1抑制RET及PLAG1的表达并影响神经细胞迁移和增殖功能。而且，分泌性蛋白SLIT2通过绑定其受体基因ROBO1参与抑制神经细胞的迁移、增殖功能。通过构建miR-218-1转基因小鼠，发现鼠肠道组织中神经节细胞数量明显减少并伴有神经纤维代偿性增生，证实miR-218-1参与先天性巨结肠的发生。. 本研究首次阐述miR-218-1及其SLIT2/ROBO1-miR-218-1-RET/PLAG1通路影响神经嵴细胞迁移和增殖功能在先天性巨结肠发生中的作用，不仅可以为先天性巨结肠的发生机制的研究提供新的观点，同时也为体内神经嵴细胞迁移、增殖功能障碍的早期干预寻找治疗靶点，为胚胎期先天性巨结肠的防治提供理论依据。