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)。这些在小鼠中通常不保守并且通常是细胞特异性的， 给机械研究提出了挑战，但也为人类健康和疾病的进步提供了重大机遇 疾病。在这里，我们迎接这一挑战，对我们优先考虑的三种非保守 lincRNA 进行询问 来自人类巨噬细胞的深度 RNAseq 和初步功能研究。巨噬细胞活化至多种 功能状态在心脏代谢疾病（CMD）中发挥着核心作用。我们假设人类 lincRNA 调节影响复杂 CMD 的巨噬细胞炎症和代谢功能。迄今为止监管 一些 lincRNA 对巨噬细胞生物学的影响已有报道，但绝大多数人类 巨噬细胞 lincRNA 尚待研究。通过RNAseq对原代人单核细胞来源的 巨噬细胞 (HMDM) 中，我们鉴定了 2,776 个多外显子 lincRNA，其中 >80% 未在小鼠中进行注释。 基于 (i) 巨噬细胞激活过程中的调节，(ii) 与 CMD 遗传信号重叠，(iii) 巨噬细胞 富集和丰度，(iv) CEBPa 和 PU.1 蛋白的 ChIP-seq 结合，以及 (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 lincRNA。从这些中，我们在这里关注三个 lincRNA，未表达 在小鼠巨噬细胞中，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，在人类动脉粥样硬化和 减弱 iNOS 表达和细胞凋亡（目标 1）。相反，RP11-184M15.1 在 M2 期间被高度诱导 巨噬细胞激活并调节 M2 表型（目标 2）。最后，RP11-472N13.3与中央关联 人类肥胖并减弱巨噬细胞 IFNg 信号传导（目标 3）。因为大规模的基因操纵 在原代人类巨噬细胞中存在限制，我们建议在 IPSDM 中进行 CRISPR/Cas9 基因编辑以追求 这些 CMD 相关 lincRNA 的功能基因组询问。主要调查结果将得到证实 HMDM 中 lincRNA 的敲低（KD）和过表达（OE）。定位、miR 结合和蛋白质 相互作用将通过RNA荧光原位杂交、MS2标记的RNA亲和纯化、 通过 QPCR 确认，并通过与质谱联用的生物素化 lincRNA 探针进行下拉，并使用 RNA 免疫沉淀。相互作用伙伴的 KD 和 OE 将通过特定的 miR 测试 lincRNA 的功能作用 或蛋白质靶点。人类疾病的相关性将通过将 lincRNA 定位到巨噬细胞来确定 冠状动脉粥样硬化和内脏脂肪，并通过检查 lincRNA 顺式调节变体， 通过 CMD 遗传数据集中的巨噬细胞等位基因特异性表达来识别。