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: 9983136
• 负责人: Muredach P Reilly
• 金额: $ 66.47万
• 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9983136
• 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; Coronary Arteriosclerosis; Coupled; Cytoplasm; Data; Data Set; Disease; Elements; Ensure; Exons; Fluorescent in Situ Hybridization; Functional disorder; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Diseases; Genome; Genomics; Guide RNA; Health; Human; Immunoprecipitation; In Vitro; Inflammation; Inflammatory; Intercistronic Region; Interleukin-4; Knowledge; Lipolysis; Macrophage Activation; Maps; Mass Spectrum Analysis; Metabolic; 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; cardiometabolism; clinically relevant; coronary lesion; data resource; functional genomics; genetic manipulation; human disease; human model; in silico; 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都在基因间区域，牵涉到调节元素，包括 长基因间非编码RNA（lincrNA）。这些通常在小鼠中不保守，通常是细胞特异性的， 提出机械研究的挑战，但也为人类健康的进步提供了重要的机会 疾病。在这里，我们将这一挑战在审讯三个未经保留的lincrnas方面我们优先考虑 从人类巨噬细胞中的深入RNASEQ和初步功能研究。巨噬细胞激活多样 功能状态在心脏代谢疾病（CMD）中起着核心作用。我们假设人类lincrnas 调节影响复杂CMD的巨噬细胞炎症和代谢功能。迄今为止的监管 已经报道了一些lincrnas对巨噬细胞生物学的影响，但绝大多数人 巨噬细胞lincrnas尚未研究。通过原代人单核细胞衍生的RNASEQ 巨噬细胞（HMDM），我们鉴定了2,776个多外年lincrnas，其中> 80％未在小鼠中注释。 基于（i）在巨噬细胞激活过程中的调制，（ii）与CMD的遗传信号重叠，（iii）巨噬细胞 Cebpa和Pu.1蛋白的富集和丰度，（iv）chip-seq结合，（v）相似的表达 人类诱导多能干细胞（HIPSC）衍生的巨噬细胞（IPSDM）的模式，我们选择了一组22 用于验证和初步翻译的lincrnas。从中，我们在这里重点放在三个lincrnas上，未表达 在小鼠巨噬细胞中，具有人类巨噬细胞功能的初步证据。 RP11-10J5.1是 在炎症M1激活过程中显着诱导的，在人动脉粥样硬化和 减弱iNOS表达和凋亡（AIM 1）。相反，M2期间RP11-184M15.1高度诱导 巨噬细胞激活并调节M2表型（AIM 2）。最后，RP11-472N13.3与中央 人类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纵 在原发性巨噬细胞中，我们建议在IPSDM中提出CRISPR/CAS9基因编辑以追求 这些与CMD相关的LincrNA的功能基因组询问。关键发现将得到证实 HMDM中LincrNA的敲低（KD）和过表达（OE）。定位，miR结合和蛋白质 相互作用将由RNA荧光原位杂交定义，MS2标记的RNA亲和力纯化， 用qPCR确认，并用生物素化的lincrna探针与质量光谱法耦合，并与 RNA免疫沉淀。相互作用伙伴的KD和OE将通过特定miR测试LincrNA的功能角色 或蛋白质靶标。人类疾病相关性将通过将lincrnas定位于巨噬细胞来确定 冠状动脉粥样硬化和内脏脂肪以及通过lincrna顺式调节变体的审问， 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。