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导致智力迟钝的主要原因。越来越多的证据表明，过度的细胞死亡是乙醇诱导的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根本性的差距。MicroRNAs （miRNAs）是最近发现的一类小的18-23核苷酸非编码RNA，与细胞凋亡相关基因的调控有关。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嵴细胞（NCCs）凋亡。我们的长期目标是针对乙醇致畸的有效策略的发展；通过靶向参与细胞凋亡的特定途径预防乙醇诱导的细胞凋亡的策略。这项特别提议的总体目标是建立miRNA作为预防乙醇诱导的细胞凋亡和致畸的可行靶点。待验证的中心假设是miR-34a和miR-125b通过调控p53和Bcl2信号通路调节乙醇诱导的NCCs细胞凋亡，抑制miR-34a或过表达miR-125b可预防乙醇诱导的畸胎化。我们的假设是在我们实验室产生的强有力的初步数据的基础上制定的。为了验证我们的假设，将解决以下具体目的：目的1：表征miR-34a和miR-125b在乙醇诱导的NCCs细胞凋亡中的作用。我们将确定乙醇对NCCs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2：验证miR-34a和miR- 125b通过调控p53和Bcl2通路调控乙醇诱导的NCCs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信号抑制中的作用，以及乙醇下调miR-125b对乙醇暴露的nc中p53和Bcl2信号的影响来实现。目的3：验证miR-34a和miR-125b的调节代表了一种预防乙醇诱导的畸胎症的新治疗策略的假设。我们将确定miR-34a的敲低或miR-125b的过表达是否会减少小鼠胚胎中乙醇诱导的畸形。这项工作是创新的，因为它侧重于一种新的方法，靶向参与细胞凋亡的mirna，以防止乙醇诱导的畸胎性。本申请中描述的理论概念也具有高度创新性，因为这是第一个试图通过新认识的miR-34a和miR-125b在细胞凋亡中的作用来特异性地阻止乙醇诱导的细胞凋亡和致畸的研究。这项研究的结果将是重要的，因为完成这些目标所获得的见解将有助于阐明mirna在调节乙醇诱导的致畸中的作用。他们还有望产生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.