microRNA-1调控神经嵴细胞发育和衍化而影响颅面发育的机制研究

Malformations involving the craniofacial regions are common birth defect found in human beings. In the craniofacial development, neural crest cells are one of the key contributors. Our previous studies on human, mouse and zebrafish revealed that over-expression of miR-1 results in no head, mandibular prognathism and reduced pigment， which indicated that miR-1 may play important roles in craniofacial development by regulating the development and derivation of neural crest cell. The objective of this study is to investigate the effects of miR-1 on neural crest cell in the craniofacial development and its underlying mechanism. Knockout and overexpression fish model will be developed to test whether miR-1 affect the proliferation, apoptosis, migration and differentiation of neural crest cells, and organs and tissues derived from neural crest cells such as head, maxillae, tooth, nerve and pigment. iTRAQ and bioinformatics prediction will be performed to identify the targets of miR-1. By overexpression or knockout of the target genes, the mechanism of miR-1 in regulating the craniofacial defects and neural crest cells abnormality will be investigated. Through this project, we will reveal the molecular mechanisms underlying regulation of zebrafish craniofacial development by microRNA-1. The results will be helpful to understand the etiologies, prevention and therapy of human craniofacial birth defects.

颅面发育畸形是一类常见的出生缺陷，神经嵴细胞是颅面发育的主要来源细胞之一。本课题组前期关于人、果蝇和斑马鱼的研究显示过表达microRNA-1(miR-1)可导致颅面萎缩、下颌前突、色素减少等畸形，表明miR-1可能通过调控神经嵴细胞发育衍化而影响颅面发育。本课题组拟进一步观测miR-1基因敲除和过表达对斑马鱼神经嵴细胞增殖、凋亡、迁移、分化的影响，以及对头颅、颌骨、牙齿、神经和色素等神经嵴细胞衍化的器官或组织的影响，并结合生物信息学和iTRAQ差异蛋白质组学研究筛选其靶基因，再进行靶基因的过表达或敲除以阐明miR-1调控神经嵴细胞的作用机制。研究结果将有助于了解miR-1对颅面发育的影响及作用机制，有望为颅面畸形症的预防和治疗提供新的靶点。

颅面发育畸形是一类常见的出生缺陷，神经嵴细胞是颅面发育的主要来源细胞之一。本课题组前期关于人、果蝇和斑马鱼的研究显示过表达microRNA-1（miR-1）可导致颅面萎缩、下颌前突、色素减少等畸形，表明miR-1可能通过调控神经嵴细胞发育衍化而影响颅面发育。本课题组进一步观测miR-1基因敲低和过表达对斑马鱼神经嵴细胞增殖、凋亡、迁移、分化的影响，以及对头颅、颌骨、牙齿、神经和色素等神经嵴细胞衍化的器官或组织的影响，并结合生物信息学和iTRAQ差异蛋白质组学研究筛选其靶基因，再进行靶基因的过表达或敲除以阐明miR-1调控神经嵴细胞的作用机制。实验中我们发现在斑马鱼体内敲低了miR-1的表达之后，神经嵴衍化来的一系列组织出现发育障碍：包括严重的下颌后缩和色素形成迟缓。通过延迟摄影技术和对神经嵴细胞迁移重要标记基因的检测，发现神经嵴细胞的迁移发生障碍。在胚胎发育至24小时，我们通过原位杂交发现神经嵴分化过程中重要的标记基因tfap2a, dlx2, dlx3b, ngn1 和 crestin的表达均存在不同程度的减少。通过蛋白质组学（iTRAQ）的研究发现，在敲低了miR-1的表达之后32个蛋白的表达出现差异，其中SEC63的上调超过1.5倍。通过荧光素酶报告基因实验证实sec63是miR-1的直接靶基因。进一步证实在敲低miR-1的同时下调sec63的表达，能够部分拯救下调miR-1所导致的发育畸形。我们的研究证实了miR-1通过调节基因sec63的表达对颅面部神经嵴正常发育产生调控作用。研究结果有助于了解miR-1对颅面发育的影响及作用机制，有望为颅面畸形症的预防和治疗提供新的靶点。