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

描述（由申请人提供）：吸毒成瘾是对公共安全和健康的根本威胁。因此，其病理生物学一直在紧张的研究。中脑核（nucleus nucleus，NAc）是一个在正常的奖赏学习和成瘾中起关键作用的大脑区域。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大脑中独特的药物特异性基因表达模式的复杂景观。虽然这种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，包括microRNA和长链非编码RNA（lncRNA），在特定的时空限制的背景下在大脑中大量表达，并且可以直接调节蛋白质编码基因。然而，对于人类大脑lncRNA在药物成瘾中的表达一无所知。该项目将填补我们对大脑基因表达和功能的认识中的这一重要空白。从成瘾和正常大脑中lncRNA表达的拟议分析中出现的见解将进一步加深我们对成瘾机制的理解，并可能促进抗成瘾疗法的发展。Specific Aim 1将构建人类lncRNA基因的综合目录，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。这是一个基本的必要性，因为没有普遍接受的参考注释的人lncRNA转录组，因为预制的微阵列存在的蛋白质编码和microRNA转录，但不是lncRNA的轮廓。具体目标2将使用目标1中创建的阵列来鉴定可卡因滥用者、海洛因滥用者和配对对照的NAc中差异表达的lncRNA，并将使用RT-PCR验证微阵列结果。结果，一个目录的lncRNA的NAc的表达被改变显着的慢性药物暴露，将提供一个基础的lncRNA的作用机制在药物成瘾的功能研究。Specific Aim 3将开始通过将公共转录因子结合位点数据集与lncRNA基因组图谱相结合来阐明差异表达lncRNA的调控，从而鉴定在特定lncRNA基因启动子处具有潜在结合位点的神经表达转录因子。将特异性lncRNA与已知的转录调控网络相匹配，将开始阐明lncRNA表达的调控，潜在地突出了可作为药物滥用治疗靶点的信号传导途径。 公共卫生相关性：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microRNA，因此被称为长链非蛋白质编码RNA（lncRNA）。以前的研究与人类药物成瘾相关的基因表达变化几乎完全集中在已知的蛋白质编码基因，这种成瘾相关的基因表达的变化，在神经回路成瘾的关键区域，仍然没有完全理解。该研究将通过将定制基因表达分析微阵列（“DNA芯片”）应用于海洛因成瘾者，可卡因成瘾者和对照核的RNA样本来测试lncRNA有助于该脑区域药物诱导的功能变化的假设，以努力分配功能和治疗相关性到那里表达的lncRNA。

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.