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，其病理生物学一直处于紧张的研究之中。伏隔核（NAc）是一个在正常的奖励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和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类别，包括microRNA和长链非编码RNA (lncRNA)，在特定的时空限制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，我们对药物成瘾过程中人脑lncRNA的表达尚不清楚。该项目将填补我们在大脑基因表达和功能方面的这一重要空白。从lncRNA在成瘾和正常大脑中的表达分析中产生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人类lncRNA转录组的参考注释，并且由于预制的微阵列存在，以分析蛋白质编码和microRNA转录物，而不是lncRNA。特异性Aim 2将使用Aim 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者NAc中差异表达的lncrna，并将使用RT-PCR验证微阵列结果。这一结果将为lncRNA在药物成瘾中的作用机制的功能研究提供基础，从而形成NAc表达因慢性药物暴露而显著改变的lncRNA目录。Specific Aim 3将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，开始阐明差异表达lncRNA的调控，从而识别在特定lncRNA基因启动子处具有潜在结合位点的神经表达转录因子。将特定的lncRNA纳入已知的转录调控网络将开始阐明lncRNA表达的调控，潜在地突出可作为药物滥用治疗靶点的信号通路。

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.