LncRNA Transcriptional Mechanisms of Coronary Artery Disease Risk

治疗性危险因素修改显著降低了冠心病(CAD) 然而，西方人群的重大风险是由于影响疾病的共同遗传遗传变异。 如果没有特定的治疗方法，人们对此仍然知之甚少。为了进一步发展我们的长期 为了确定这种遗传风险的分子基础，我们参与了全基因组的 确定与冠状动脉疾病(CAD)风险相关的等位基因变异的关联研究，以及这些 努力已经产生了数百个相关的基因座。然而，大多数已识别的因果变异存在于 在蛋白质编码外显子之外，在基因组中知之甚少的调节区，以及进一步 需要努力了解这种联系的机制，从而了解疾病风险。我们的中心假设 是疾病等位基因变异的一个重要子集，主要调控长非编码rna(Lncrna)。 表达，这种效应通过功能调节因果蛋白编码基因(Pcgene)的表达 基因组相互作用，如染色体环路。我们在这里的目标是研究这些lncRNA在 参与调节CAD因果pcGenes的表达及其实现机制 功能。我们的理论基础是lncRNAs在遗传和表观遗传之间起着关键的中介作用。 阐明它们的作用机制是了解冠心病风险的一个关键方面。为了获得 关于这些分子在CAD中的作用模式的基本信息，我们建议 研究人冠状动脉平滑肌细胞(HCASMC)中的lncRNA。在目标1中，我们将识别lncRNA 在这些细胞中受疾病相关刺激的调节，并映射到CAD GWAs基因座。共表达网络 分析将这些lncRNA连接到pcGenes，并启动网络和途径分析，以开始 建立它们的生物功能关联。在目标2中，我们将定位表达数量性状基因座变异 (EQTL)针对每个LncRNA，使用高通量等位基因特异性表达方法，该方法提供 低丰度RNA的定量。将对发现的lncRNA eQTL进行调查，以确定 它们与CAD GWAs因果变异以及基因组分子性状QTL共定位。CRISPR基因组 将使用编辑来验证eQTL，并确认pcgene身份。在目标3中，我们将采用CRISPR 抑制和单细胞RNA测序(PerturSeq)绘制受 疾病相关的lncRNAs，并研究其对HCASMC的体外细胞作用。这些研究将是 在我们对原代培养的HCASMC进行大量工作的帮助下，HCASMC表征了表观基因组修饰、染色质 可及性和循环，以及我们为绘制CAD GWA因果变体和基因而做出的努力，这些变量和基因介导了 此单元格类型。这项工作具有很高的创新性，因为它结合了在高度 与疾病相关的细胞类型和意义，因为它将整合lncRNA，它们的调节变异，以及 从分子机制上探讨冠心病风险的病因。

Therapeutic risk factor modification has provided a significant decrease in coronary artery disease (CAD) in Western populations, however, significant risk is due to common inherited genetic variation that affects disease pathways in the vessel wall and remains poorly understood without specific therapies. To further our long-term goal of characterizing the molecular basis for this genetic risk, we have participated in genome-wide association studies (GWAS) identifying allelic variation linked to coronary artery disease (CAD) risk, and these efforts have yielded hundreds of associated loci. However, the majority of identified causal variation resides outside of protein coding exons, in regulatory regions of the genome that are poorly understood, and further efforts are required to understand the mechanisms of association and thus disease risk. Our central hypothesis is that an important subset of disease allelic variation primarily regulates long non-coding RNA (lncRNA) expression, with this effect modulating causal protein coding gene (pcGene) expression through functional genomic interactions such as chromosomal looping. Our objective here is to investigate the role these lncRNAs play in mediating expression of CAD causal pcGenes, and the mechanism by which they accomplish this function. Our rationale is that lncRNAs serve as a critical intermediary between genetic and epigenetic signaling, and that elucidating their mechanism of function is a key aspect of understanding CAD risk. To gain fundamental information regarding the mode of action of these molecules in the context of CAD, we propose to study human coronary artery smooth muscle cell (HCASMC) lncRNAs. In Aim 1, we will identify lncRNAs regulated in these cells by disease-related stimuli and that map to CAD GWAS loci. Co-expression network analyses will connect these lncRNAs to pcGenes, and initiate network and pathway analyses to begin to establish their biological functional associations. In Aim 2, we will map expression quantitative trait loci variants (eQTLs) for each of the lncRNAs, using a high-throughput allele-specific expression method that provides quantification of low abundance RNAs. Discovered lncRNA eQTLs will be investigated to determine whether they colocalize with CAD GWAS causal variation, as well as genomic molecular trait QTLs. CRISPR genome editing will be employed to validate the eQTLs, and confirm pcGene identity. In Aim 3, we will employ CRISPR inhibition and single cell RNA sequencing (PerturbSeq) to map the transcriptional networks regulated by the disease related lncRNAs, and also investigate their in vitro cellular effects on HCASMC. These studies will be aided by our extensive work with primary cultured HCASMC characterizing epigenome modification, chromatin accessibility, and looping, and our efforts to map CAD GWAS causal variants and genes that mediate risk in this cell type. This work is highly innovative in that it combines unique genomic datasets developed in a highly disease relevant cell type and significant since it will integrate lncRNAs, their regulatory variation, and molecular mechanisms into the etiology of CAD risk.