Brain microRNA-mRNA regulatory networks and alcohol use disorders

项目摘要/摘要 酒精使用障碍(AUD)的特征是强迫性和无节制的饮酒，导致社交 和职业损伤。虽然基因变异会增加患AUDS的风险，但长期饮酒 饮酒可以独立地导致对酒精的耐受和依赖。然而，目前还不清楚是如何 长期饮酒会改变基因表达，并导致AUDS潜在的神经适应。的确有 有证据表明，调控靶基因(或mRNA)表达的小非编码microRNAs(MiRNAs) 转录后水平，在大脑中含量丰富，并在各种生物过程中发挥重要作用 如神经元分化、突触形成和可塑性。此外，每个miRNA都可以调节 许多不同的靶mRNAs和每个mRNAs的表达可以被不同的miRNAs靶向。 因此，调节对酒精反应的mRNAs表达的大脑miRNAs可能作用于酒精的上游- 诱导的神经适应。拟议研究的目的是识别失调的miRNAs及其 靶向AUD患者大脑中的mRNAs并产生与AUD相关的miRNA-mRNA调节 网络。中心假说是AUD相关的miRNAs与奖赏相关的靶mRNAs相互作用 大脑区域，形成对AUD发育至关重要的miRNA-mRNA调控网络。这 将通过追求三个具体目标来检验假设：(1)确定差异表达的miRNAs和 至少八个奖赏相关脑区(前额叶皮质、伏隔核、腹侧被盖)的mRNAs AUD受试者的脑区、海马体、杏仁核、壳核、尾状核和小脑) 测序(miRNA-Seq和mRNA-Seq)；(2)产生AUD相关的miRNA-mRNA调控网络 使用综合生物信息学分析与奖励相关的大脑区域；以及(3)提炼与AUD相关的miRNA- 通过使用实验方法验证预测的miRNA-mRNA对， 例如在细胞系中高通量3‘UTR报告分析和在人类中的miRNA-mRNA相互作用分析 胚胎干细胞(HESC)来源的GABA能皮质神经元(作为体外细胞模型)。建议数 这项研究具有重要意义，因为它将识别和验证AUD相关的miRNA-mRNA调控网络 在奖赏相关的大脑区域，从而提高了我们对AUDS表观遗传机制的理解。这个 拟议的研究具有创新性，因为先进技术(如下一代测序、状态- 用于构建miRNA-mRNA网络的最先进的生物信息学计划，高通量3‘非编码区报告 将使用干细胞来源的神经元中的miRNA-mRNA相互作用模型)。我们的长期目标 是通过靶向特定的miRNAs及其靶基因来开发治疗AUDS的新药物。

PROJECT SUMMARY/ABSTRACT Alcohol use disorders (AUDs) are characterized by compulsive and uncontrolled alcohol use, leading to social and occupational impairments. While genetic variation can result in an increased risk of AUDs, chronic alcohol consumption can independently lead to alcohol tolerance and dependence. However, it is unknown how chronic alcohol consumption alters gene expression and leads to neuroadaptations underlying AUDs. There is evidence that small noncoding microRNAs (miRNAs), which regulate target gene (or mRNA) expression at the posttranscriptional level, are abundant in the brain and play important roles in a variety of biological processes such as neuronal differentiation and synapse formation and plasticity. Additionally, each miRNA can regulate the expression of a number of different target mRNAs and each mRNA can be targeted by different miRNAs. Thus, brain miRNAs that regulate the expression of alcohol-responsive mRNAs may act upstream of alcohol- induced neuroadaptations. The objective of the proposed study is to identify dysregulated miRNAs and their target mRNAs in the brains of AUD subjects and generate AUD-associated miRNA-mRNA regulatory networks. The central hypothesis is that AUD-associated miRNAs interact with target mRNAs in reward-related brain regions, forming miRNA-mRNA regulatory networks that are critical for AUD development. This hypothesis will be tested by pursuing three specific aims: (1) identify differentially expressed miRNAs and mRNAs in at least eight reward-related brain regions (prefrontal cortex, nucleus accumbens, ventral tegmental area, hippocampus, amygdala, putamen, caudate, and cerebellum) of AUD subjects using next-generation sequencing (miRNA-Seq and mRNA-Seq); (2) generate AUD-associated miRNA-mRNA regulatory networks in reward-related brain regions using integrated bioinformatics analyses; and (3) refine AUD-associated miRNA- mRNA regulatory networks by validating the predicted miRNA-mRNA pairs using experimental approaches, such as high throughput 3' UTR reporter assays in cell lines and miRNA-mRNA interaction analyses in human embryonic stem cell (hESC)-derived GABAergic cortical neurons (as an in vitro cellular model). The proposed research is significant because it will identify and validate AUD-associated miRNA-mRNA regulatory networks in reward-related brain regions, thus improving our understanding of the epigenetic mechanisms of AUDs. The proposed research is innovative because advanced technologies (such as next-generation sequencing, state- of-the-art bioinformatics programs for miRNA-mRNA network construction, high-throughput 3' UTR reporter assay, and miRNA-mRNA interaction modeling in stem cell-derived neurons) will be used. Our long-term goal is to develop novel pharmacological treatment for AUDs by targeting specific miRNAs and their target genes.