Computational and functional strategies to decipher lncRNAs in human atherosclerosis

该提案解决了细胞特异性功能和长期非编码疾病病因方面的知识空白。 RNA（lncRNA）在人类动脉粥样硬化中的作用尽管在心血管疾病中有功能性lncRNA的突出例子， 由于它们缺乏保守性和细胞特异性，限制了我们对它们在CVD中作用的理解。 这些挑战在人类动脉粥样硬化中尤其成问题，其特征在于复杂的多- 细胞损伤此外，最近的单细胞（sc）RNAseq数据，包括我们的初步研究表明， 相对于mRNA，在原代人细胞中的lncRNA表达可能限于关键细胞亚群。一个 最重要的假设是，许多人类lncRNA通过它们的表达调节动脉粥样硬化和CVD风险。 在特定病变细胞亚群中的离散表达和功能。因此，更准确地了解 lncRNA与人类动脉粥样硬化的细胞特异性关系是驱动基于机制的临床研究所必需的。 翻译.在这里，我们解决了人类动脉粥样硬化和CVD风险中lncRNA的关键问题。首先， lncRNA在人类病变中表达并与临床CVD相关？第二，对于表达于 人类病变，在哪些特定的病变细胞亚群是他们的功能？在目标1中，我们将解决第一个问题 通过大型巢式病例对照的深度RNAseq分析lncRNA的差异表达 （n=260例“有症状/不稳定”斑块和n=260例“无症状/稳定”斑块） 动脉粥样硬化，来自慕尼黑血管生物库（MVB）。我们还将确定lncRNA是否表现出 有症状/不稳定斑块与无症状/稳定斑块之间的差异等位基因特异性表达（ASE）， 如果具有差异ASE的lncRNA的顺式eQTL变体与冠心病（CHD）相关， 公共基因数据集优先化的lncRNA将在人类中进行细胞特异性功能基因组随访 血管细胞，包括我们的人诱导多能干细胞（hIPSC）血管模型。在目标2中，我们建议 使用一种新的去卷积算法，并将来自Aim 1的大规模批量RNAseq数据与选择性 新鲜病变的单细胞（sc）RNAseq（n=60），以鉴定与肿瘤相关的亚群及其lncRNA。 症状性/不稳定斑块，与CHD有因果遗传关系。新鲜颈动脉的ScRNAseq 损伤将用于聚集细胞并鉴定损伤亚群。分析结果将允许 所有MVB本体RNAseq病变的细胞亚群组成的计算去卷积（n=520），以及 亚群特异性lncRNA表达的分配以及与有症状/不稳定斑块的关系。 亚群特异性lncRNA顺式eQTL也将被鉴定并用于确定它们的因果关系 CHD的基因数据。这些发现，加上亚群特异性功能研究，将定义 亚群特异性lncRNA在人类动脉粥样硬化中的功能我们的提案利用了独特的基因组 数据，创新的计算方法和功能基因组学，以及跨学科的专业知识，以指导体内 在动脉粥样硬化CVD中细胞特异性血管lncRNA功能的翻译和精确治疗靶向。

This proposal addresses knowledge gaps in cell-specific function and disease causation of long non-coding RNAs (lncRNAs) in human atherosclerosis. Despite prominent examples of functional lncRNAs in cardiovascular diseases (CVD), their lack of conservation and cell-specificity have limited our understanding of their role in CVD. These challenges are particularly problematic in human atherosclerosis which is characterized by complex multi- cellular lesions. Further, recent single cell (sc)RNAseq data including our preliminary studies suggest that, relative to mRNAs, lncRNA expression in primary human cells may be restricted to key cell subpopulations. An overarching hypothesis is that many human lncRNAs modulate atherosclerosis and CVD risk via their discrete expression and function in specific lesion cell subpopulations. Thus, more precise knowledge of lncRNA cell-specific relationship to human atherosclerosis is required to drive mechanism-based clinical translation. Here we address key questions for lncRNAs in human atherosclerosis and CVD risk. First, which lncRNAs are expressed in human lesions and associate with clinical CVD? Second, for lncRNAs expressed in human lesions, in which specific lesion cell subpopulation are they functional? In Aim 1, we will address the first issue by analyzing differential expression of lncRNAs through deep RNAseq of a large nested case-control (n=260 with “symptomatic/unstable” and n=260 with “asymptomatic/stable” plaques) study of carotid atherosclerosis from the Munich Vascular Biobank (MVB). We will also determine whether lncRNAs demonstrate differential allele specific expression (ASE) between symptomatic/unstable vs. asymptomatic/stable plaques and if cis-eQTL variants for lncRNAs with differential ASE are associated with coronary heart disease (CHD) in large public genetic datasets. Prioritized lncRNAs will undergo cell-specific functional genomic follow-up in human vascular cells including our human induced pluripotent stem cell (hIPSC) vascular models. In Aim 2, we propose to use a novel deconvolution algorithm and integration of large-scale bulk RNAseq data from Aim 1 with selective single cell (sc)RNAseq of fresh lesions (n=60) to identify subpopulations and their lncRNAs that associate with symptomatic/unstable plaques and have causal genetic relationships to CHD. ScRNAseq of the fresh carotid lesions will be used to cluster cells and identify lesion subpopulations. Result from this analysis will permit computational deconvolution of the cell subpopulation composition of all MVB bulk RNAseq lesions (n=520) and assignment of subpopulation-specific lncRNA expression and relationship to symptomatic/unstable plaques. Subpopulation-specific lncRNA cis-eQTLs also will be identified and used to determine their causal relationship to CHD in genetic datasets. These findings, coupled to subpopulation-specific functional studies, will define subpopulation-specific lncRNA functions in human atherosclerosis. Our proposal leverages unique genomic data, innovative computational methods and functional genomics, and interdisciplinary expertise to direct in vivo translation and precision therapeutic targeting of cell-specific vascular lncRNA functions in atherosclerotic CVD.