Human LincRNAs in Macrophage Biology and Related Cardiometabolic Diseases

大多数人类复杂的性状遗传信号存在于基因间隔区，涉及调控元件，包括 长的基因间非编码RNA(LincRNAs)。它们通常在小鼠中不保守，通常是细胞特异性的， 对机械学研究提出了挑战，但也为人类健康和 疾病。在这里，我们接受了这一挑战，询问了我们优先考虑的三个非保守的lincRNA 来自深层RNAseq和人类巨噬细胞的初步功能研究。巨噬细胞活化到多种 功能状态在心脏代谢性疾病(CMD)中起着核心作用。我们假设人类的LincRNA 调节巨噬细胞炎症和代谢功能，从而影响复杂的CMD。到目前为止的监管 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 巨噬细胞的lincRNAs还有待研究。通过原代人单核细胞来源的RNAseq 巨噬细胞(HMDM)，我们鉴定了2,776个多外显子连接RNA，其中80%在小鼠中没有注释。 基于(I)巨噬细胞激活期间的调节，(Ii)与CMD的遗传信号重叠，(Iii)巨噬细胞 (4)CEBPA和PU.1蛋白的ChIP-seq结合；(V)相似表达 在人类诱导多能干细胞(HiPSC)来源的巨噬细胞(IPSDM)中，我们选择了一组22 用于验证和初步翻译的lincRNA。在此，我们关注三个未表达的lincRNAs 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 在炎性M1激活过程中显著诱导，在人类动脉粥样硬化和 减少iNOS表达和细胞凋亡(目标1)。相反，RP11-184M15.1在M2期间高度诱导 巨噬细胞激活和调节M2表型(目标2)。最后，RP11-472N13.3与中央 人类肥胖和减弱巨噬细胞IFNG信号(目标3)。因为大规模的基因操作 在原代人巨噬细胞中是有限的，我们建议在IPSDM中进行CRISPR/Cas9基因编辑来追求 这些与CMD相关的lincRNAs的功能基因组询问。关键发现将得到以下方面的证实 HMDM中lincRNAs的敲除(KD)和过表达(OE)。定位、miR结合与蛋白质 相互作用将通过RNA荧光原位杂交、MS2标记的RNA亲和纯化、 用定量聚合酶链式反应确认，并用生物素化lincRNA探针与质谱仪和 RNA免疫沉淀。互动伙伴的KD和OE将通过特定的miR测试lincRNAs的功能角色 或蛋白质靶标(S)。人类疾病的相关性将通过在巨噬细胞中定位lincRNAs来确定 冠状动脉粥样硬化和内脏脂肪以及通过询问lincRNA顺式调节变异体， 在CMD基因数据集中，通过巨噬细胞等位基因的特异性表达进行鉴定。

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.