miRNA contributes to epigenetic regulation of NR2B gene during ethanol withdrawal

描述（由申请人提供）：NMDA受体上调是一个主要的神经适应过程，在慢性乙醇暴露戒断后引起兴奋综合征。NMDA受体在戒断期间的神经适应机制尚不清楚，但一些证据表明基因表达的改变起着重要作用。MicroRNAs （miRNAs）是最近发现的一类小的非编码rna，在各种生物过程中参与基因表达的微调。我们最近发现了miRNA在慢性间歇乙醇暴露下的独特表达模式，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在酒精戒断期间NR2B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MeCP2来调节DNA甲基化，并随后参与NR2B基因的神经适应性上调。特异性目的1：在体外鉴定并验证miR-152、-150和-126靶向MeCP2并调控NR2B基因的DNA甲基化。我们将证实这些miRNAs(1)利用慢病毒传递系统将这些miRNAs引入培养的神经元，与MeCP2 mRNA的3' UTR发生生物相互作用；(2)利用体外报告基因检测功能抑制目标努力水平（抑制）；(3)利用硫酸氢盐焦磷酸测序确定这些mirna对NR2B基因DNA甲基化的调控作用。特异性目的2：研究这些mirna是否参与乙醇依赖小鼠乙醇戒断诱导的适应调节。TH-和GAD67-GFP转基因小鼠将以CIE方案暴露于酒精中，然后停药5天。通过激光捕获显微解剖获得小鼠中脑多巴胺神经元VTA中的DAergic或GABAergic神经元，并用于分析这些mirna的差异表达，MeCP2和NR2B基因的mRNA以及NR2B启动子的DNA甲基化。本研究将确定mirna参与小鼠乙醇戒断过程中NR2B基因表观遗传调控的新机制。首次将激光捕获显微解剖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.