Differential Expression and Regulatory Functions of Long Non-Coding RNA Molecules

描述(申请人提供)：吸毒是对公共安全和健康的根本威胁。因此，它的病理生物学一直处于紧张的研究之中。伏隔核(NAC)是大脑的一个区域，在正常的奖赏学习和成瘾过程中发挥着关键作用。基因表达微阵列已被用于阐明成瘾中NAC神经可塑性的分子基础，揭示了动物模型和人类死后脑中不同的和药物特异性的基因表达模式的复杂图景。虽然这类微阵列主要测量蛋白质编码基因的mRNA水平，但最近的主要研究表明，非蛋白质编码RNA类别，包括microRNA和长非编码RNA(LncRNA)，在特定的时空受限环境中在大脑中大量表达，并可以直接调节蛋白质编码基因。然而，关于人类大脑中的lncRNA在药物成瘾中的表达还知之甚少。拟议中的项目将填补我们对大脑基因表达和功能知识的这一重要空白。通过对成瘾者和正常大脑中lncRNA表达的分析，将有助于我们对成瘾机制的理解，并可能有助于抗成瘾疗法的发展。具体目标1将构建一个全面的人类LncRNA基因目录，并将在该目录的基础上设计定制的全基因组LncRNA表达分析微阵列。这是一种基本的必要性，因为没有普遍接受的人类lncRNA转录组的参考注释，而且由于存在预制微阵列来分析蛋白质编码和microRNA转录本，而不是lncRNA。专门的目标2将使用在目标1中创建的阵列来识别在可卡因滥用者、海洛因滥用者和配对配对的对照组的NAC中差异表达的lncRNAs，并将使用RT-PCR验证微阵列结果。这一结果将为研究LncRNA在药物成瘾中的作用机制提供一个功能研究的基础。具体目标3将通过结合公共转录因子结合位点数据集和lncRNA基因组图谱，开始阐明差异表达的lncRNAs的调控，从而确定神经元表达的转录因子与特定lncRNA基因启动子的潜在结合位点。将特定的lncRNAs装配到已知的转录调控网络中，将开始阐明lncRNA表达的调控，潜在地突出适合作为药物滥用治疗靶点的信号通路。 与公共卫生相关：一些重要的功能调节RNA分子既不是蛋白质编码基因的信使RNA，也不是microRNAs，因此被称为长非蛋白质编码RNAs(Long Non-Protein-Coding RNAs，LncRNAs)。以前对与人类药物成瘾相关的基因表达变化的研究几乎完全集中在已知的蛋白质编码基因上，而伏隔核--成瘾神经回路中的关键区域--基因表达的这种与成瘾相关的变化仍然不完全清楚。这项拟议的研究将通过对海洛因成瘾者、可卡因成瘾者和对照伏核RNA样本应用定制基因表达分析微阵列(DNA芯片)，努力分配那里表达的lncRNAs的功能和治疗相关性，来检验lncRNAs有助于药物诱导的这一大脑区域功能变化的假设。

DESCRIPTION (provided by applicant): Drug addiction is a fundamental threat to public safety and health. Its pathobiology has therefore been under intense study. The nucleus accumbens (NAc) is a brain region that plays a key role in normal reward learning and in addiction. Gene expression microarrays have been employed to elucidate the molecular basis of NAc neuroplasticity in addiction, revealing a complex landscape of distinct and drug-specific gene expression patterns in animal models and human postmortem brain. While such microarrays predominantly measure mRNA levels of protein coding genes, major recent studies demonstrate that non-protein-coding RNA classes, including microRNA and long noncoding RNA (lncRNA), are abundantly expressed in the brain within specific spatiotemporally restricted contexts, and can directly regulate protein-coding genes. Nevertheless, nothing is known about human brain lncRNA expression in drug addiction. The proposed project will fill this important gap in our knowledge of brain gene expression and function. Insights emergent from the proposed analysis of lncRNA expression in the addicted and normal brain will further our understanding of addiction mechanisms and may facilitate the development of anti-addiction therapies. Specific Aim 1 will construct a comprehensive catalog of human lncRNA genes, and will build upon that catalog to design a custom genome-wide lncRNA expression analysis microarray. This is a fundamental necessity, since there is no universally accepted reference annotation of the human lncRNA transcriptome, and since prefabricated microarrays exist to profile protein coding and microRNA transcripts but not lncRNA. Specific Aim 2 will use the arrays created in Aim 1 to identify lncRNAs differentially expressed in the NAc of cocaine abusers, heroin abusers, and pair-matched controls, and will validate the microarray results using RT-PCR. The outcome, a catalog of lncRNAs whose NAc expression is altered significantly by chronic drug exposure, will provide a foundation for functional studies of lncRNA action mechanisms in drug addiction. Specific Aim 3 will begin to elucidate the regulation of the differentially expressed lncRNAs by combining public transcription factor binding site datasets with lncRNA genomic mappings, thereby identifying neurally-expressed transcription factors with potential binding sites at promoters of specific lncRNA genes. Fitting specific lncRNAs into known transcription regulatory networks will begin to elucidate the regulation of lncRNA expression, potentially highlighting signaling pathways amenable as therapy targets for drug abuse. PUBLIC HEALTH RELEVANCE: Some important functional regulatory RNA molecules are neither messenger RNAs of protein coding genes nor microRNAs, and have therefore been termed long non-protein-coding RNAs (lncRNAs). Previous studies of gene expression changes associated with human drug addiction have focused almost exclusively on known protein-coding genes, and such addiction-related changes in gene expression in the nucleus accumbens, a pivotal region in the neural circuitry of addiction, remain incompletely understood. The proposed study will test the hypothesis that lncRNAs contribute to drug-induced functional changes in this brain region, by applying custom gene expression analysis microarrays ("DNA chips") to heroin-addict, cocaine-addict, and control nucleus accumbens RNA samples in an effort to assign function and therapeutic relevance to lncRNAs expressed there.