miRNA contributes to epigenetic regulation of NR2B gene during ethanol withdrawal

乙醇戒断期间 miRNA 有助于 NR2B 基因的表观遗传调控

• 批准号: 8734304
• 负责人: ALAN FRAZER
• 金额: $ 20.85万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734304
• 关键词: 3' Untranslated Regions; Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcohol withdrawal syndrome; Alcoholism; Alcohols; Binding; Biological; Biological Assay; Biological Process; Breathing; Cells; Chronic; DNA; DNA Methylation; Dependence; Development; Disease; Epigenetic Process; Ethanol; Ethanol dependence; Excision; Functional RNA; Gene Expression; Genes; Genetic Transcription; Genomic DNA; Goals; Health; Health Care Costs; In Vitro; Lead; Mediating; Messenger RNA; Methyl-CpG-Binding Protein 2; MicroRNAs; Midbrain structure; Molecular; Mus; N-Methyl-D-Aspartate Receptors; Neurons; Pattern; Phenotype; Play; Population; Prevention strategy; Process; Receptor Up-Regulation; Regimen; Regulation; Regulatory Element; Reporter Genes; Repression; Role; Sampling; Site; Substance Withdrawal Syndrome; Syndrome; System; Therapeutic; Time; Transgenic Mice; Up-Regulation; Withdrawal; Withdrawal Symptom; Work; alcohol exposure; alcohol relapse; alcoholism therapy; base; cell type; demethylation; design; dopaminergic neuron; gamma-Aminobutyric Acid; in vivo; innovation; insight; laser capture microdissection; neuroadaptation; new therapeutic target; novel; promoter; public health relevance; pyrosequencing; response

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.

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，在退出慢性乙醇暴露后会导致兴奋性综合征。戒断期间NMDA受体神经适应的机制尚不清楚，但是几条证据表明，基因表达改变起着重要作用。 MicroRNA（miRNA）是最近发现的一类小型非编码RNA，与各种生物学过程中基因表达的微调有关。我们最近在小鼠vta da Neuron中对慢性间歇性乙醇暴露的反应显示了miRNA的独特表达模式，包括miR -152，-150和-126的上调节。这项拟议的研究旨在研究miRNA在戒酒过程中NR2B基因的神经适应性上调的DNA甲基化中的作用。因此，我们假设酒精戒断引起的miR-152，-150和-126的上调通过下调MECP2来调节DNA甲基化，并随后参与NR2B基因的神经适应性上调。具体目标1：确定并验证miR -152，-150和-126靶MECP2，并在体外调节NR2B基因的DNA甲基化。我们将使用慢病毒递送系统与MECP2 mRNA的3'UTR进行生物学相互作用，以确认这些miRNA（1）将这些miRNA引入培养的神经元中； （2）使用体外报告基因测定法抑制目标努力水平（抑制）； （3）确定这些miRNA使用硫酸硫酸盐硫酸盐测序的调节作用在NR2B基因的DNA甲基化方面的调节作用。具体目的2：研究miRNA是否参与了乙醇依赖小鼠乙醇戒断诱导的适应的调节。 TH-和GAD67-GFP转基因小鼠将通过CIE疗法接触酒精，然后戒断5天。将使用激光捕获显微解剖学获得中脑多巴胺神经元VTA中的DAERGIC或GABA能神经元，用于分析这些miRNA，MECP2和NR2B基因的mRNA的差异表达，以及NR2B促进剂的DNA甲基化。拟议的研究将确定一种新的机制，即miRNA在小鼠乙醇戒断期间有助于NR2B基因的表观遗传调节。通过激光捕获显微解剖获得的单个神经元种群将首次用于研究与酒精戒断相关的神经适应的表观遗传机制。