Role of microRNAs in Ethanol-induced Apoptosis and Teratogenesis

描述(由申请人提供)：产前酒精暴露是导致智力低下的主要已知原因。越来越多的证据表明，细胞过度死亡是酒精所致出生缺陷发病机制的主要组成部分。然而，在理解乙醇如何导致胚胎细胞凋亡方面存在着根本性的差距。MicroRNAs(MiRNAs)是最近发现的一类18-23个核苷酸的非编码小RNA，参与了细胞凋亡相关基因的调控。我们最近发现，促凋亡miRNA miR-34a和抗凋亡miRNA miR-125b参与了乙醇诱导的神经脊细胞(NCC)的凋亡。我们的长期目标是开发有效的策略来对抗乙醇的致畸作用；这些策略的基础是通过靶向参与细胞凋亡的特定途径来预防酒精诱导的细胞凋亡。这一建议的总体目标是将miRNA确立为预防乙醇诱导的细胞凋亡和致畸的可行靶点。有待检验的中心假设是miR-34a和miR-125b通过调控p53和bcl2信号通路来调控乙醇诱导的神经干细胞凋亡，抑制miR-34a或过表达miR-125b可以防止乙醇诱导的畸形。我们的假设是基于我们实验室提供的强有力的初步数据而提出的。为了验证我们的假设，将解决以下具体目标：目的：研究miR-34a和miR-125b在乙醇诱导NCC细胞凋亡中的作用。我们将确定乙醇对神经干细胞和小鼠胚胎中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作用。目的：验证miR-34a和miR-125b通过调控p53和bcl2通路调控乙醇诱导神经干细胞凋亡的假说。这将通过确定miR-34a在乙醇诱导的P53激活和Bcl2信号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信号的影响来实现。目的：验证miR-34a和miR-125b的调控是预防乙醇致畸的一种新的治疗策略的假设。我们将确定miR-34a的敲除或miR-125b的过度表达是否能减少乙醇诱导的小鼠胚胎畸形。这项拟议的工作是创新的，因为它专注于一种新的方法，针对参与细胞凋亡的miRNAs，以防止乙醇诱导的畸形。本申请中描述的理论概念也具有很高的创新性，因为这是第一项试图通过新发现的miR-34a和miR-125b在细胞凋亡中的作用来防止乙醇诱导的细胞凋亡和畸形发生的研究。这项研究的结果将是重要的，因为这些目标的实现所获得的洞察力将有助于阐明miRNAs在调节乙醇诱导的畸形发生中的作用。预计他们还将提出防止乙醇致畸的策略，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.