miRNA contributes to epigenetic regulation of NR2B gene during ethanol withdrawal

描述（由申请方提供）：NMDA受体上调是一种主要的神经适应性过程，在从长期乙醇暴露中戒断后会导致兴奋性综合征。NMDA受体在戒断过程中的神经适应机制尚不清楚，但一些证据表明，基因表达的变化起着重要作用。microRNAs（miRNAs）是近年来发现的一类参与基因表达调控的小分子非编码RNA。我们最近显示了响应于慢性间歇性乙醇暴露的miRNA的独特表达模式，包括小鼠VTA DA神经元中miR-152、-150和-126的上调。本研究旨在研究miRNA在酒精戒断过程中NR 2B基因神经适应性上调的DNA甲基化中的作用。因此，我们假设酒精戒断诱导的miR-152、-150和-126上调通过下调MeCP 2调节DNA甲基化，随后参与NR 2B基因的神经适应性上调。具体目的1：在体外鉴定并验证miR-152、-150和-126靶向MeCP 2并调节NR 2B基因的DNA甲基化。我们将证实这些miRNAs（1）与MeCP 2 mRNA的3' UTR生物学相互作用，使用慢病毒递送系统将这些miRNAs引入培养的神经元中;（2）使用体外报告基因测定在功能上抑制靶向作用水平（抑制）;和（3）使用硫酸氢盐焦磷酸测序确定这些miRNAs对NR 2B基因的DNA甲基化的调节作用。具体目的2：研究miRNAs是否参与乙醇依赖小鼠乙醇戒断诱导的适应性调节。TH-和GAD 67-GFP转基因小鼠将以CIE方案暴露于酒精，然后停药5天。使用激光捕获显微切割获得小鼠中脑多巴胺神经元的VTA中的DA能或GABA能神经元，并用于分析这些miRNA、MeCP 2和NR 2B基因的mRNA的差异表达以及NR 2B启动子的DNA甲基化。这项研究将确定一种新的机制，即miRNAs有助于小鼠乙醇戒断过程中NR 2B基因的表观遗传调控。首次将激光捕获显微切割获得的单个神经元群体用于研究酒精戒断相关神经适应的表观遗传机制。

DESCRIPTION (provided by applicant): NMDA receptor up-regulation is a major neuroadaptive process that causes excitatory syndrome upon withdrawal from chronic ethanol exposure. The mechanisms of neuroadaptation of NMDA receptors during withdrawal remain unclear, but several lines of evidence suggest that changed gene expression plays important roles. MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs which are involved in the fine tuning of gene expression in various biological processes. We have recently shown a distinct expression pattern of miRNA in response to chronic intermittent ethanol exposure, including the up- regulation of miR-152, -150, and -126 in mice VTA DA neuron. This proposed study is designed to study the role of miRNA in the DNA methylation underlying neuroadaptive up-regulation of NR2B gene during alcohol withdrawal. Therefore, we hypothesize that alcohol withdrawal-induced up-regulation of miR-152, -150 and - 126 modulates DNA methylation by down-regulating MeCP2 and are subsequently involved in neuroadaptive up-regulation of the NR2B gene. Specific Aim 1: Identify and validate that miR-152, -150 and -126 target MeCP2 and regulate DNA methylation of the NR2B gene in vitro. We will confirm these miRNAs (1) biologically interact with the 3' UTR of MeCP2 mRNA using a lentiviral delivery system to introduce these miRNAs into cultured neurons; (2) functionally inhibit the target effort level (repression) using an in vitro reporter gene assay; and (3) determie the regulatory role of these miRNAs on DNA methylation of the NR2B gene using bisulfate pyrosequencing. Specific Aim 2: To investigate whether the miRNAs are involved in the regulation of ethanol withdrawal-induced adaptation in ethanol dependence mice. TH- and GAD67-GFP transgenic mice will be exposed to alcohol with a CIE regimen followed by 5 days withdrawal. The DAergic or GABAergic neurons in the VTA of midbrain dopamine neurons in the mice will be obtained using laser capture microdissection and used for the analysis of the differential expression of these miRNAs, mRNA of MeCP2 and NR2B genes as well as DNA methylation of the NR2B promoter. The proposed study will determine a novel mechanism that miRNAs contribute to the epigenetic regulation of the NR2B gene during ethanol withdrawal of mice. For the first time, single neuronal populations obtained by laser capture microdissection will be used to study epigenetic mechanism on alcohol withdrawal-related neuroadaptation.