Human LincRNAs in Macrophage Biology and Related Cardiometabolic Diseases

巨噬细胞生物学和相关心脏代谢疾病中的人类 LincRNA

• 批准号: 9402855
• 负责人: Muredach P Reilly
• 金额: $ 72.21万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9402855
• 关键词: Adipose tissue; Affinity Chromatography; Alleles; Antisense Oligonucleotides; Apoptosis; Atherosclerosis; Attenuated; Autophagocytosis; Binding; Binding Proteins; Bioinformatics; Biological Assay; Biology; CRISPR interference; CRISPR/Cas technology; Cell Nucleus; Cells; Central obesity; ChIP-seq; Chromatin; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Complex Genetic Trait; Computer Simulation; Coronary; Coronary Arteriosclerosis; Coupled; Cytoplasm; Data; Data Set; Disease; Elements; Ensure; Exons; Fluorescent in Situ Hybridization; Functional disorder; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic; Genome; Genomics; Guide RNA; Health; Hereditary Disease; Human; Immunoprecipitation; In Vitro; Inflammation; Inflammatory; Intercistronic Region; Interleukin-4; Knowledge; Lesion; Lipolysis; Macrophage Activation; Maps; Mass Spectrum Analysis; Metabolic; Metabolic Diseases; Modeling; Molecular; Mus; Myelogenous; Obesity; Oxidative Phosphorylation; Pattern; Phagocytosis; Phenotype; Play; Protein Isoforms; Proteins; RNA; RNA Probes; RNA purification; Regulatory Element; Reporting; Repression; Reproducibility; Research; Role; Seeds; Signal Transduction; Testing; Translations; Untranslated RNA; Validation; Variant; Visceral; Work; atheroprotective; base; cardiometabolic risk; clinically relevant; data resource; disorder risk; functional genomics; genetic manipulation; human disease; induced pluripotent stem cell; inducible gene expression; innovation; knock-down; macrophage; monocyte; new therapeutic target; novel; novel diagnostics; overexpression; transcriptome sequencing; translational study

Most human complex trait genetic signals lie in intergenic regions, implicating regulatory elements, including long intergenic non-coding RNAs (lincRNAs). These are typically not conserved in mouse and often cell-specific, raising challenges to mechanistic study but also providing a major opportunity for advances in human health and disease. Here, we embrace this challenge in interrogation of three non-conserved lincRNAs that we prioritized from deep RNAseq and preliminary functional studies in human macrophages. Macrophage activation to diverse functional states plays a central role in cardiometabolic diseases (CMD). We hypothesize that human lincRNAs modulate macrophage inflammatory and metabolic functions that impact complex CMD. To date regulatory effects of a few lincRNAs on macrophage biology have been reported, but the vast majority of human macrophage lincRNAs has yet to be studied. Through RNAseq of primary human monocyte-derived macrophages (HMDM), we identified 2,776 multi-exon lincRNAs of which >80% are not annotated in mouse. Based on (i) modulation during macrophage activation, (ii) overlap with genetic signals for CMD, (iii) macrophage enrichment and abundance, (iv) ChIP-seq binding of CEBPa and PU.1 proteins, and (v) similar expression pattern in human induced pluripotent stem cells (hiPSC) derived macrophages (IPSDM), we selected a set of 22 lincRNAs for validation and preliminary translation. From these, we focus here on three lincRNAs, not expressed in mouse macrophages, that have preliminary evidence of human macrophage functions. RP11-10J5.1 is markedly induced during inflammatory M1 activation, has increased expression in human atherosclerosis and attenuates iNOS expression and apoptosis (Aim 1). In contrast, RP11-184M15.1 is highly induced during M2 macrophage activation and modulates M2 phenotype (Aim 2). Finally, RP11-472N13.3 associates with central human obesity and attenuates macrophage IFNg signaling (Aim 3). Because large-scale genetic manipulation in primary human macrophages is limiting, we propose CRISPR/Cas9 gene-editing in IPSDM to pursue functional genomic interrogations of these CMD-relevant lincRNAs. Key findings will be corroborated by knockdown (KD) and overexpression (OE) of lincRNAs in HMDM. Localization, miR binding and protein interactions will be defined by RNA fluorescence in situ hybridization, MS2-tagged RNA affinity purification, confirmed with QPCR, and pull-down with biotinylated lincRNA probes coupled to mass-spectrometry and with RNA immunoprecipitation. KD and OE of interacting partners will test lincRNAs functional roles via specific miR or proteins target(s). Human disease relevance will be determined by localizing lincRNAs to macrophages in coronary atherosclerosis and visceral adipose and through interrogation of lincRNA cis-regulatory variants, identified via macrophage allele specific expression, in CMD genetic datasets.

大多数人类复杂性状遗传信号位于基因间区域，涉及调控元件，包括 长基因间非编码RNA（lincRNA）。这些通常在小鼠中不保守，并且通常是细胞特异性的， 对机制研究提出了挑战，但也为人类健康的进步提供了重大机遇， 疾病在这里，我们接受了这一挑战，在审问三个非保守的lincRNA，我们优先考虑 来自人类巨噬细胞的深度RNAseq和初步功能研究。巨噬细胞活化， 功能状态在心脏代谢疾病（CMD）中起核心作用。我们假设人类lincRNA 调节巨噬细胞炎症和代谢功能，影响复杂的CMD。迄今为止的监管 已经报道了一些lincRNA对巨噬细胞生物学的影响，但绝大多数人的lincRNA对巨噬细胞生物学的影响是不明显的。 巨噬细胞lincRNA还有待研究。通过原代人单核细胞源性 在HMDM巨噬细胞中，我们鉴定了2，776个多外显子lincRNA，其中>80%在小鼠中未注释。 基于（i）巨噬细胞活化期间的调节，（ii）与CMD的遗传信号重叠，（iii）巨噬细胞 1蛋白的ChIP-seq结合，以及（v）相似的表达 为了研究人诱导多能干细胞（hiPSC）衍生的巨噬细胞（IPSDM）的模式，我们选择了一组22 lincRNA用于验证和初步翻译。从这些，我们在这里集中在三个lincRNA，不表达 在小鼠巨噬细胞中，有初步证据表明人类巨噬细胞功能。RP 11 -10J5.1是 在炎性M1活化过程中显著诱导，在人动脉粥样硬化中表达增加， 减弱iNOS表达和细胞凋亡（Aim 1）。相反，RP 11 - 184 M15.1在M2期间高度诱导 巨噬细胞活化并调节M2表型（Aim 2）。最后，RP 11 -472N13.3与中心关联 人肥胖和减弱巨噬细胞IFNg信号传导（Aim 3）。因为大规模的基因操作 在原代人类巨噬细胞中的CRISPR/Cas9基因编辑是有限的，我们建议在IPSDM中进行CRISPR/Cas9基因编辑， 这些CMD相关lincRNA的功能基因组询问。主要调查结果将得到以下方面的证实： HMDM中lincRNA的敲低（KD）和过表达（OE）。定位、miR结合和蛋白质 通过RNA荧光原位杂交，MS 2标记的RNA亲和纯化， 用QPCR确认，并用与质谱偶联的生物素化lincRNA探针和与 RNA免疫沉淀。相互作用伴侣的KD和OE将通过特异性miR测试lincRNA的功能作用 或蛋白质靶标。人类疾病相关性将通过将lincRNA定位于巨噬细胞来确定。 冠状动脉粥样硬化和内脏脂肪，并通过询问lincRNA顺式调节变体， 通过巨噬细胞等位基因特异性表达在CMD遗传数据集中鉴定。