miR-200s通过靶向调控p38信号通路介导酒精诱导神经嵴细胞凋亡参与胎儿酒精谱系障碍发生的机制研究

Fetal Alcohol Spectrum Disorders, caused by prenatal alcohol exposure, is among the most devastating birth defects. Growing evidence suggests that excessive neural crest cells death is a critical event of the pathogenesis of ethanol-induced birth defects. However, there is a fundamental gap in understanding how ethanol leads to apoptotic cells death in embryos. Our recent findings demonstrated that up-regulation of the expression levels of p38 and its specific upstream kinase MKK6 plays a critical role in ethanol-induced apoptosis. Our preliminary studies using bioinformatics tools showed that p38 and its upstream activator MKK6 are potential targets of miR-200a and miR-200c, respectively, and that overexpression of miR-200s can prevent neural crest cells against ethanol-induced apoptosis. On the basis of the current findings, we propose that regulation of p38 signaling by miR-200s is a critical molecular event in ethanol-induced teratogenesis. Our current application aims to investigate the mechanisms by which miR-200s regulate ethanol-induced apoptosis through targeting of p38 signaling on a cellular level, with help of molecular technologies et al., and to elucidate the embryo protective role of miR-200s in ethanol-induced teratogenesis, with assistance of cutting edge technologies, such as whole embryo culture, ultra-sound guided embryo visualization, and microinjection et al. The proposed studies will not only fundamentally advance the molecular pathogenesis of FASD but also contribute to providing us a novel target and a new strategy for intervention and early diagnosis of FASD.

孕期酒精暴露引起的胎儿酒精谱系障碍是最具破坏性的出生缺陷疾病之一。神经嵴细胞群过度凋亡是酒精致畸的重要特征，但相关机制仍不明确。我们新近研究发现：p38和其上游特异性激酶MKK6表达水平的上调在酒精诱导凋亡中扮演重要角色。经生物信息学预测p38和MKK6分别是miR-200a和miR-200c的潜在作用靶点且前期实验发现上调miR-200s可抵抗酒精诱导的凋亡损伤。据此推测miR-200s调控p38信号是酒精致畸的重要分子事件。此次申请旨在前期基础上利用分子生物学等技术在细胞水平上深入研究酒精通过miR-200s调控p38信号的机制；并借助胚胎体外培养、超声波生物显微镜和胚胎显微注射等技术在动物模型上证实miR-200s在酒精致畸中的生物学功能及机制并初步探讨其在胚胎保护中的角色。该项目的成功实施有助于加深对酒精致畸分子机制的认识，也将为酒精致畸的早期诊断和防治提供新靶点及新策略。

酒精被认为是一种致畸剂，它是引起子代智力发育迟缓的最主要非遗传性因素之一。酒精相关致畸机制研究的开展，有助于寻找出生缺陷发病机制的新靶点并加深对相关信号通路功能的认识，突破性成果有望为阻断胎儿畸形的发生提供新策略，是亟需深入探索的研究领域。本项目综合运用了分子生物学、细胞生物学、和胚胎模型工具，基于表型到机制的理论思想。研究揭示p38 MAPK参与酒精诱导神经嵴细胞的凋亡，miR-200a的上调可通过直接靶向p38 MAPK来预防酒精诱导的细胞凋亡；研究发现MKK6在酒精诱导p38活化和凋亡中起着关键作用，并且miR-200c的过表达可以通过直接靶向MKK6抑制p38信号传导来减少酒精诱导的细胞凋亡；研究确定西兰花提取物SFN可以表观遗传的形式恢复细胞凋亡关键调控基因Bcl2的表达并通过消除酒精引起的Bcl2启动子区域的高甲基化作用来减弱酒精诱导的凋亡过程；研究首次证明胚胎早期酒精暴露可以显著降低化学诱导幼虫斑马鱼癫痫发作的阈值并增加幼虫斑马鱼癫痫发作的严重程度。以上研究不仅有助于加深对酒精致畸分子机制的认识，也为酒精致畸的早期诊断和防治提供了新靶点及新策略。