lncRNA-MEG3靶向调控RAC1信号通路在先天性巨结肠中的作用及机制研究

先天性巨结肠（HSCR)是遗传和环境多因素导致肠神经嵴细胞增殖、分化和迁移障碍的先天性疾病，目前机制不清。现有研究表明lncRNA可通过影响神经细胞的生物学功能参与HSCR发生，但其分子机制不明。我们前期研究发现HSCR患儿存在差异表达的lncRNA-MEG3，可影响神经母细胞瘤细胞增殖迁移和凋亡，但具体调控通路及机制不详，目前通过靶基因预测发现MEG3与RAC1存在靶向调控关系。因此，本项目在此基础上以HSCR患儿结肠组织及神经母细胞瘤细胞为研究对象，应用FISH及CHIP实验观察MEG3与RAC1的分布及结合情况，通过建立MEG3过表达细胞模型应用双素荧光素酶实验鉴定RAC1是MEG3靶基因，并在细胞模型中调控MEG3及RAC1通路，观察其基因及蛋白的变化和对细胞生物学功能的影响，阐明MEG3对RAC1通路的调控作用，旨在为探索HSCR发病的分子机制及寻找治疗靶点提供新的思路。

Hirschsprung's disease(HSCR) is a congenital disease caused by multiple factors including genetic and environmental factors that regulate enteric neural crest cells (ENCCs) proliferation, differentiation and migration,but the pathogenesis is unclear.Though long non-coding RNAs (lncRNA) have been expound to be involved in the appearance of HSCR by affecting the biological function of nerve cells in present study,its biological mechanism in the development of HSCR still remains unknown.The previous studies of our team found that lncRNA-MEG3 is abnormally expressed in samples from HSCR,and contributed to the neuroblastoma Cells proliferation and apoptosis,but the regulatory mechanism is unknown.Recently,We found 3΄UTR of Rac1 as the putative target gene for MEG3 by nucleotide blast prediction of MEG3.Therefore,combining with previous reports and online targeting predicting resources,the colonic tissues of children with HSCR and neuroblastoma cell lines were used as the research objects,applies FISH and CHIP experiments to observe the distribution and combination of MEG3 and RAC1,and takes the dual-luciferase reporter assay to validate RAC1 as target gene of MEG3 in the MEG3 overexpressed cell model, and regulates MEG3 and RAC1 signal pathways in the cell models, observes changes of signal pathway-related genes and proteins and their effects on cell biological functions, clarifies the regulatory effect and molecular mechanism of MEG3 on RAC1 pathway, and provides new ideas for HSCR pathogenesis and finding therapeutic targets.