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性过程，在退出长期乙醇暴露后会导致兴奋综合征。 NMDA 受体在戒断过程中的神经适应机制仍不清楚，但一些证据表明基因表达的改变发挥着重要作用。 MicroRNA (miRNA) 是最近发现的一类小非编码 RNA，参与各种生物过程中基因表达的微调。我们最近显示了 miRNA 响应慢性间歇性乙醇暴露的独特表达模式，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 NR2B 基因神经适应性上调 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 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：在体外鉴定并验证 miR-152、-150 和 -126 靶向 MeCP2 并调节 NR2B 基因的 DNA 甲基化。我们将确认这些 miRNA (1) 使用慢病毒递送系统将这些 miRNA 引入培养的神经元中，与 MeCP2 mRNA 的 3' UTR 发生生物学相互作用； (2) 使用体外报告基因测定功能性抑制目标努力水平（抑制）； (3) 使用硫酸氢盐焦磷酸测序确定这些 miRNA 对 NR2B 基因 DNA 甲基化的调节作用。具体目标 2：研究 miRNA 是否参与乙醇依赖小鼠乙醇戒断诱导适应的调节。 TH-和GAD67-GFP转基因小鼠将按照CIE方案暴露于酒精，然后停药5天。通过激光捕获显微切割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。拟议的研究将确定 miRNA 在小鼠乙醇戒断过程中对 NR2B 基因进行表观遗传调控的新机制。通过激光捕获显微切割获得的单个神经元群将首次用于研究酒精戒断相关神经适应的表观遗传机制。