SDF-1/CXCR4轴通过下调miR-9多靶向诱导上皮间质转化调控神经嵴干细胞迁移增殖与存活的研究

The enteric nervous system (ENS) disease is one of the most common congenital gastrointestinal malformations in children, occurring in 1/3000 live births.The physiological basis of ENS disease is poorly understood although neural crest stem cells (NCSCs) migration, proliferation and colonization in mesenchyme has been shown involved. It has been found in the last project（National Natural Scientific Foundation 30700917）that expression of CXCR4 protein was abnormal in Hirschspring disease, and ENS was malformed along with upregulation of miR-9 when the CXCR4 gene was knockout. These results indicate SDF-1/CXCR4 axis and miR-9 play a critical role in ENS formation. In order to verify this hypothesis, in this study, the murine model organism will be used. In the mouse embryos, the temporal expression of SDF-1/CXCR4 proteins was firstly elaborated by double immune fluorescent staining. NCSCs were isolated,cultured and treated by means of RNAi, the expression levels of miR-9, E-cadherin and other EMT derivants such as Snail2, ZEB1 and ZEB2 were carried out along with Transwell, MTT and BrdU trials to verify the role of SDF-1/CXCR4 axis in NCSCs migration, proliferation and apoptosis in vitro through induction of epithelia-mesenchymal transition. The target downregulation of EMT derivants Snail2, ZEB1 and ZEB2 by miR-9 was also observed and verified. CXCR4（＋）and CXCR4（－）NCSCs were both labeled with different fluorescent material and injected into age-matched allergenic murine embryos(including the transgenic mice in which CXCR4 was knockout) by means of microinjection. The NCSCs migration, proliferation and apoptosis in vivo were then observed to verify the roles of SDF-1/CXCR4 axis in ENS formation. In this way, we demonstrate that SDF-1/CXCR4 axis controls migration, proliferation and survival of neural crest stem cells through induction of miR-9 mediated epithelia-mesenchymal transition.Results from this set can provide a strong evidence for SDF-1/CXCR4 and miR-9 to be used as a valuable biomarker and novel target in ENS disease in the future.

肠神经系统（ENS）发育异常是最常见的消化道畸形之一，严重影响我国出生人口质量, 但ENS发育的调控机制尚不清楚。我们前期研究（国家自然科学基金30700917）通过比较蛋白质组学发现CXCR4蛋白在先天性巨结肠中异常表达，将CXCR4基因敲除后发现小鼠的miR-9表达上调且ENS发育异常，说明CXCR4和miR-9参与ENS发育调控，但具体机制不清楚。本研究拟从胚胎，细胞，动物和临床水平观察SDF-1/CXCR4轴对miR-9的调控作用，理论预测miR-9与EMT关键分子的结合位点并验证miR-9的靶向沉默作用，证实SDF-1/CXCR4轴通过PI3K/AKT和ERK途径下调miR-9多靶向诱导EMT调控神经嵴干细胞迁移增殖与存活，探讨SDF-1/CXCR4轴的调控机制。SDF-1/CXCR4和miR-9分子有望成为ENS发育异常诊断的新型分子标记，为ENS疾病的治疗提供新的靶点和思路。

肠神经系统（ENS）发育异常是最常见的消化道畸形之一，严重影响我国出生人口质量, 但ENS发育的调控机制尚不清楚。我们前期研究（国家自然科学基金30700917）通过比较蛋白质组学发现CXCR4蛋白在先天性巨结肠中异常表达，将CXCR4基因敲除后发现小鼠的microRNAs表达异常且ENS发育异常，说明CXCR4和microRNAs参与ENS发育调控，但具体机制不清楚。本研究从胚胎，细胞，动物和临床水平观察SDF-1/CXCR4轴对microRNAs的调控作用，理论预测microRNAs与EMT关键分子的结合位点并验证靶向沉默作用，证实SDF-1/CXCR4轴多靶向诱导EMT调控神经嵴干细胞迁移增殖与存活，探讨SDF-1/CXCR4轴的调控机制；同时课题还对ENS发育异常的形态标记以及无创诊断进行探讨，证实外周血Sox10启动子区域甲基化以及ZEB2的点突变可以作为ENS疾病无创诊断的分子标记；本项课题现如期进行，过程顺利，现已发表论文8篇，其中SCI收录5篇，同时本课题培养1名博士和4名硕士研究生，在后续的试验中拟再培养博士1名，硕士2名，发表SCI论文至少2篇。