Role of microRNAs in Ethanol-induced Apoptosis and Teratogenesis

描述（由申请人提供）：产前乙醇暴露是已知的智力低下的主要原因。越来越多的证据表明，过度细胞死亡是乙醇引起的出生缺陷发病机制的主要组成部分。然而，对于乙醇如何导致胚胎细胞凋亡的理解存在根本性的差距。 MicroRNA (miRNA) 是最近发现的一类小型 18-23 核苷酸非编码 RNA，与细胞凋亡相关基因的调节有关。我们最近发现，促凋亡 miRNA miR-34a 和抗凋亡 miRNA miR-125b 参与乙醇诱导的神经嵴细胞 (NCC) 细胞凋亡。我们的长期目标是制定针对乙醇致畸的有效策略；基于通过靶向参与细胞凋亡的特定途径来预防乙醇诱导的细胞凋亡的策略。这一特定提案的总体目标是将 miRNA 确立为预防乙醇诱导的细胞凋亡和畸胎发生的可行靶标。待测试的中心假设是 miR-34a 和 miR-125b 通过调节 p53 和 Bcl2 信号通路来调节乙醇诱导的 NCC 细胞凋亡，并且抑制 miR-34a 或过度表达 miR-125b 可以预防乙醇诱导的致畸。我们的假设是根据我们实验室产生的强有力的初步数据制定的。为了检验我们的假设，将解决以下具体目标： 目标 1：表征 miR-34a 和 miR-125b 在乙醇诱导的 NCC 细胞凋亡中的作用。我们将确定乙醇对 NCC 和小鼠胚胎中 miR-34a 和 miR-125b 表达的影响，以及 miR-34a 和 miR-125b 在乙醇诱导的细胞凋亡中的参与。目的 2：检验 miR-34a 和 miR-125b 通过调节 p53 和 Bcl2 通路调节乙醇诱导的 NCC 细胞凋亡的假设。这将通过确定 miR-34a 在乙醇诱导的 p53 激活和 Bcl2 信号传导抑制中的作用以及乙醇下调 miR-125b 对乙醇暴露的 NCC 中 p53 和 Bcl2 信号传导的影响来实现。目标 3：检验 miR-34a 和 miR-125b 的调节代表了预防乙醇诱导的畸胎发生的新治疗策略这一假设。我们将确定 miR-34a 的敲低或 miR-125b 的过表达是否会减少乙醇诱导的小鼠胚胎畸形。这项工作具有创新性，因为它专注于一种新方法，针对参与细胞凋亡的 miRNA，以防止乙醇诱导的畸胎发生。该申请中描述的理论概念也具有高度创新性，因为这是第一项尝试特别通过新近认识的 miR-34a 和 miR-125b 在细胞凋亡中的作用来预防乙醇诱导的细胞凋亡和致畸的研究。这项研究的结果将是意义重大的，因为通过实现这些目标所获得的见解将有助于阐明 miRNA 在调节乙醇诱导的畸胎发生中的作用。他们还有望制定预防乙醇致畸的策略，并从根本上推进 FASD 研究领域。

DESCRIPTION (provided by applicant): Prenatal ethanol exposure is the leading known cause of mental retardation. Growing evidence suggests that excessive cell death is a major component of the pathogenesis of ethanol-induced birth defects. However, there is a fundamental gap in understanding how ethanol leads to apoptotic cell death in embryos. MicroRNAs (miRNAs) are a recently discovered class of small 18-23 nucleotide non-coding RNA that have been implicated in the regulation of genes involved in apoptosis. We have recently discovered that miR-34a, a pro-apoptotic miRNA and miR-125b, an anti-apoptotic miRNA are involved in ethanol-induced apoptosis in neural crest cells (NCCs). Our long-term goal is directed toward the development of effective strategies against ethanol's teratogenesis; strategies based on prevention of ethanol-induced apoptosis through targeting specific pathways involved in apoptosis. The overall objective of this particular proposal is to establish miRNA as a feasible target for the prevention of ethanol-induced apoptosis and teratogenesis. The central hypothesis to be tested is that miR-34a and miR-125b modulate ethanol-induced apoptosis in NCCs by the regulation of p53 and Bcl2 signaling pathways and that the inhibition of miR-34a or overexpression of miR-125b can prevent ethanol-induced teratogenesis. Our hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory. To test our hypothesis, the following specific aims will be addressed: Aim1: To characterize the role of miR-34a and miR-125b in ethanol-induced apoptosis in NCCs. We will determine the effects of ethanol on miR-34a and miR-125b expression in NCCs and in mouse embryos, and the involvement of miR-34a and miR-125b in ethanol-induced apoptosis. Aim2: To test the hypothesis that miR-34a and miR- 125b modulate ethanol-induced apoptosis in NCCs by the regulation of p53 and Bcl2 pathways. This will be accomplished by determining the role of miR-34a in ethanol-induced activation of p53 and suppression of Bcl2 signaling and the effects of down-regulation of miR-125b by ethanol on p53 and Bcl2 signaling in ethanol- exposed NCCs. Aim3: To test the hypothesis that modulation of miR-34a and miR-125b represents a novel therapeutic strategy for preventing ethanol-induced teratogenesis. We will determine whether knockdown of miR-34a or overexpression of miR-125b diminishes ethanol-induced malformations in mouse embryos. The proposed work is innovative, because it focuses on a novel approach, targeting miRNAs involved in apoptosis, to preventing ethanol-induced teratogenesis. The theoretical concept described in this application is also highly innovative because this is the first study attempting to prevent ethanol-induced apoptosis and teratogenesis specifically through the newly recognized actions of miR-34a and miR-125b in apoptosis. The results from this study will be significant, because the insights gained by the accomplishment of these aims will help in elucidating the role of miRNAs in modulating ethanol-induced teratogenesis. They are also expected to yield strategies for preventing ethanol's teratogenesis and to fundamentally advance the field of FASD research.