miRNA in cerebrospinal fluid in CFS

摘要：“CFS患者脑脊液中的miRNA” 外切小体是膜结合的微囊(图1)图1.外切体的形成。 从细胞中释放出来，并在细胞间传递 短(22-25个核苷酸)、非编码、消息抑制RNA (MiRNAs)、基因组DNA、蛋白质和膜脂。这个 胞外体表面有CD9、CD63、CD81等多种配体 与内皮和其他表面的整合素和其他受体结合 目标单元格。外切体和靶细胞膜融合在一起 MiRNAs被释放到细胞质中。促炎作用 包括转化生长因子-？转运和分泌 一氧化氮、活性氧化剂、IL-1？和IL-18。外切体 可能作为循环中的激素或病毒样炎性小体 在大脑等器官中传递损伤信号。之前和之后- 突触神经元和神经胶质细胞有活跃的外体系统，有助于神经可塑性，但也 运输淀粉样蛋白、tau蛋白和Pron蛋白以及狂犬病病毒。 MiRNAs是分散在基因组中的短基因的产物。它们绑定到精确的 选定的mRNA中的序列。这种结合阻止了靶基因的翻译并阻止了蛋白质的合成。 综合。超过1500个人类miRNAs以细胞特有的方式调节大约50%的已知蛋白质。 外体miRNAs可以通过阻止表达来启动、扩大和维持功能障碍的表型 目标细胞中的关键蛋白质。MiRNAs可能是衰老、轻度认知障碍、 阿尔茨海默氏症、多发性硬化症、心肌病和其他疾病。在人类突触中，10个miRNAs调节 242个突触前蛋白和304个突触后蛋白中的80%。脑脊液混合标本的分析 流体通过下一代RNA测序(NGS)鉴定出133个独特的miRNAs。然而，NGS目前 对单个受试者的miRNAs模式的研究效率低下。这是因为miRNA的产量是 低到每个受试者可能需要2毫升的脑脊液，或者样本混合在一起。 研究对象。因此，我们建议使用miRNAs的定量聚合酶链式反应来研究脑脊液。 外体蛋白包括与RNA结合的热休克蛋白和其他细胞质蛋白；细胞表面 CD9、CD63、CD81和人类白细胞抗原G等受体，以及其他跨膜蛋白。这些可能会揭示出 分泌外切体的细胞类型的性质。 初步数据。慢性疲劳综合症(CFS)和海湾战争疾病(GWI)退伍军人也会见了 CFS标准显示了显著的、特定的、中枢神经系统功能障碍。GWI子集具有 已被确认身份。MiRNA参与了这些GWI亚集的形成。这表明CFS表型是客观定义的。 也可以通过miRNA模式(“生物签名”)发现和区分。外切体RNA、蛋白质和 可以从新鲜和-80°C冷冻的脑脊液中提纯脂质成分。对脑脊液进行NGS检查 来自静息状态的静坐对照(SC)受试者和由他们的磁感应强度定义的两组GWI受试者 磁共振成像和运动反应。这3个群体都有独特的miRNA图谱。MiR22有 应激试验与可逆性心动过速(START)和应激试验的等值水平 起源于幻觉(STOP)的标本。仅在SC中发现11个miRNAs。START有7分 独特的miRNAs，STOP有2个独特的miRNAs。这些模式可能为深入了解 运动性不适、疼痛、自主神经和认知功能障碍以及特定表型 生物标志物。这些来自GWI和CFS的数据提示CFS的表型和特定的miRNAs生物标记物。 目标。像GWI一样，CFS是一种异质性疾病，具有可以识别的潜在表型 来自脑脊液外切体中表达的miRNAs的特定组合。这一独特的假设 假设CFs的子集具有特定的脑细胞miRNAs模式。外切小体的不同模式 MiRNA和蛋白的表达将区分CFS和SC，并为诊断提供生物标志物。 Exosome蛋白可识别突触前、突触后、小胶质细胞、星形胶质细胞或少突胶质细胞 起源。靶向外体生物学的药物可能对CFS或选定的CFS表型有益。 特异性目的1：分离脑脊液外切体。使用定量聚合酶链式反应(Q- 聚合酶链式反应)以确定380个miRNAs在单个CFs和SC受试者中表达前后的模式 锻炼身体。确定区分CFS和SC的模式，并识别CFS受试者的潜在表型。 表征miRNA调节的蛋白质，并推断其对CFS脑生物学的影响。 特异性目的2：分离脑脊液外切体并纯化其蛋白。执行体量 外体蛋白蛋白质组学鉴定的光谱分析。确定潜在的污染源 基于这些蛋白质的外体，以及基于外体表面性质的潜在靶细胞 受体、跨膜蛋白和囊泡内蛋白。

ABSTRACT: "miRNA in CSF of CFS" Exosomes are membrane-bound microvesicles (Figure 1) Figure 1. Exosome formation. that are released from cells and mediate cell-to-cell transfer of short (22-25 nucleotides), noncoding, message inhibitory RNAs (miRNAs), genomic DNA, proteins and membrane lipids. The exosome surface has CD9, CD63, CD81 and other ligands that bind to integrins and other receptors on endothelium and other target cells. The exosome and target cell membranes fuse and miRNAs are released into the cytoplasm. Proinflammatory roles include transport of transforming growth factor-ß, and secretion of nitric oxide, reactive oxidant species, IL-1ß and IL-18. Exosomes may act as circulating hormone- or virus-like inflammasomes to transmit injury signals in organs such as the brain. Pre- and post- synaptic neurons and glia have active exosome systems that contribute to neural plasticity, but that also transport amyloid, tau and prion proteins and rabies viruses. miRNAs are products of short genes that are scattered throughout the genome. They bind to precise sequences in selected mRNAs. The binding prevents translation of the target mRNA and prevents protein synthesis. Over 1500 human miRNAs regulate about 50% of known proteins in cell-specific fashion. Exosome miRNAs may initiate, expand, and maintain dysfunctional phenotypes by preventing the expression of critical proteins in targeted cells. miRNAs may be biomarkers of aging, mild cognitive impairment, Alzheimer's, multiple sclerosis, cardiomyopathy, and other diseases. In human synapses, ten miRNAs regulate 80% of the 242 presynaptic and 304 post-synaptic proteins. Analysis of pooled specimens of cerebrospinal fluid by next generation RNA sequencing (NGS) identified 133 unique miRNAs. However, NGS is currently inefficient for studying the patterns of miRNAs from individual subjects. This is because the yield of miRNA is low so that >2 ml of cerebrospinal fluid per individual subject may be needed, or the samples pooled between subjects. Therefore, we propose to use quantitative PCR of miRNAs for this cerebrospinal fluid study. Exosome proteins include RNA-binding heat shock and other cytoplasmic proteins; cell surface receptors such as CD9, CD63, CD81 and HLA-G; and other transmembrane proteins. These may reveal the nature of the cell type that secreted the exosome. Preliminary Data. Chronic Fatigue Syndrome (CFS) and Gulf War Illness (GWI) veterans who also met CFS criteria have demonstrated significant, specific, central nervous system dysfunction. GWI subsets have been identified. miRNA contributes to these GWI subsets. This suggests objectively defined CFS phenotypes may also be discovered and discriminated by miRNA patterns ("biosignatures"). Exosome RNA, protein, and lipid components can be purified from fresh and -80°C frozen CSF. NGS was performed on cerebrospinal fluid from sedentary control (SC) subjects at rest and two subsets of GWI subjects defined by their magnetic resonance imaging and responses to exercise. The 3 groups had unique miRNA patterns. MIR22 had equivalent levels in SC and the Stress Test Associated Reversible Tachycardia (START) and Stress Test Originated Phantom Perception (STOPP) specimens. Eleven miRNAs were found in only in SC. START had 7 unique miRNAs, and STOPP had 2 unique miRNAs. These patterns may provide insights into mechanisms of exercise-induced malaise, pain, autonomic and cognitive dysfunction as well as being phenotype - specific biomarkers. These data from GWI with CFS suggest phenotypes and specific miRNAs biomarkers in CFS. Objective. Like GWI, CFS is a heterogenous disorder with latent phenotypes that can be identified from specific combinations of miRNAs expressed in cerebrospinal fluid exosomes. This unique hypothesis presumes that subsets of CFS have specific patterns of brain cell miRNAs. Distinct patterns of exosome miRNA and protein expression will discern CFS subjects from SC, and provide biomarkers for diagnosis. Exosome proteins may identify presynaptic, postsynaptic, microglial, astrocyte, or oligodendrocyte cells of origin. Drugs that target exosome biology may be beneficial in CFS or selected CFS phenotypes. SPECIFIC AIM 1: Isolate cerebrospinal fluid exosomes. Use quantitative polymerase chain reaction (Q- PCR) to identify the patterns of 380 miRNAs expressed in individual CFS and SC subjects before and after exercise. Determine patterns that discriminate CFS from SC, and identify latent phenotypes of CFS subjects. Characterize the miRNA-regulated proteins and infer the consequences for CFS brain biology. SPECIFIC AIM 2: Isolate cerebrospinal fluid exosomes and purify their proteins. Perform mass spectrometry for proteomic identification of exosome proteins. Determine the potential sources of the exosomes based on these proteins, and potential target cells based on the nature of the exosome surface receptors, transmembrane and intravesicular proteins.