Computational and functional strategies to decipher lncRNAs in human atherosclerosis

这项建议解决了长时间非编码的细胞特定功能和疾病原因方面的知识空白 人类动脉粥样硬化中的RNA(LncRNAs)。尽管心血管中有功能的lncRNAs的突出例子 疾病(CVD)，它们缺乏保守性和细胞特异性，限制了我们对它们在CVD中作用的了解。 这些挑战在人类动脉粥样硬化中尤其成问题，动脉粥样硬化的特点是复杂的多 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mRNAs，LncRNA在原代人类细胞中的表达可能仅限于关键细胞亚群。一个 最重要的假设是，许多人类lncRNA通过其基因调控动脉粥样硬化和心血管疾病的风险。 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细胞与人类动脉粥样硬化的特异性关系是驱动基于机制的临床所必需的 翻译。在这里，我们讨论了lncRNAs在人类动脉粥样硬化和心血管疾病风险中的关键问题。首先，哪一个 LncRNAs在人类皮损中表达，并与临床CVD有关？第二，对于在 人类病变，它们在哪些特定的病变细胞亚群中起作用？在目标1中，我们将解决第一个问题 一个大型嵌套病例对照的深层RNAseq分析LncRNAs差异表达问题 (260例“有症状/不稳定”和260例“无症状/稳定”斑块)颈动脉研究 来自慕尼黑血管生物库(MVB)的动脉粥样硬化。我们还将确定lncRNAs是否显示 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) 具有不同酶活性的lncRNAs的cis-eQTL变异是否与大量的冠心病(CHD)相关 公开的基因数据集。优先选择的lncRNAs将在人类体内进行细胞特异性功能基因组研究 血管细胞，包括我们的人类诱导多能干细胞(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和集成来自目标1的大规模RNAseq数据和选择性 新鲜皮损(n=60)的单细胞(Sc)RNAseq，以识别与以下相关的亚群及其LncRNA 有症状/不稳定斑块，与冠心病有因果遗传关系。新鲜颈动脉的ScRNAseq 病变将被用来聚集细胞和识别病变亚群。这一分析的结果将允许 所有MVB Bulk RNAseq病变(n=520)细胞亚群组成的计算去卷积 亚群特异性lncRNA的表达及其与症状性/不稳定斑块的关系。 亚群特异性的lncRNA cis-eQTL也将被识别并用于确定它们之间的因果关系 在基因数据集中显示为CHD。这些发现，再加上亚群特有的功能研究，将定义 亚群特异性的lncRNA在人类动脉粥样硬化中的作用。我们的方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，以及指导体内研究的跨学科专业知识 动脉粥样硬化性心血管疾病中细胞特异性血管lncRNA功能的翻译和精确靶向治疗。

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.