Impact of circadian rhythm in obtaining reference profiles of exRNAs in healthy i

描述(由申请人提供)： 摘要基于细胞外小泡(EV)是生物信息的细胞间传输者的发现，最近出现了一种新的旁分泌信号传递模式。EV介导的信号转导对细胞、组织和器官之间的代谢合作和协调至关重要，是对传统信号转导模式的补充。在此背景下，胞外RNA(ExRNA)，包括microRNAs、环状RNAs和长非编码RNA或LncRNAs，既包含在EVS中，也包含在与脂蛋白的复合体中，已被我们和其他人证明在各种细胞类型中调节基因表达和改变细胞功能。此外，在癌症等病理条件下，EVS和exRNA的数量和组成会发生变化，从而改变宿主的免疫反应，并使继发性肿瘤的行为同步。EVS和exRNAs在正常和病理条件下的分离和分析对于了解EVS和exRNAs的生物发生及其效应器功能至关重要。这些信息是新的疾病诊断和预后战略、基于生物标记物的疾病进展、治疗效果和复发监测的关键前提。EVS生物发生的一个重要决定因素是补体系统，它在激活时产生膜靶向复合体MACs(膜攻击复合体，C5b-9)，它代表未门控的钙离子和水通道。补体激活后，靶细胞胞外区和胞内区的细胞膜含有MAC，在此过程中产生大量的EVS。目前，EVS作为与细胞间信号转导和疾病诊断相关的生物实体的研究是基于这样的假设：EVS和exRNA的生物发生以稳定的速度发生，主要受宿主的健康/疾病状态的影响。我们的初步数据强烈表明，情况可能并非如此。我们的结果表明，用标准技术分离的EVS的组织来源、数量、蛋白质和RNA组成不仅取决于采血方法，还取决于采集血样的时间。在这一应用中，我们建议建立一个细胞外RNA的质量、数量和组成的基线，考虑到以下因素引起的潜在伪影：1)采血方法，2)采集时间，3)体力活动。我们用新鲜样本得到的结果，将 使用临床研究中心和输血医学部(NIH血库)存储的血浆样本进行交叉验证。

DESCRIPTION (provided by applicant): Abstract A novel paradigm in paracrine signaling has recently emerged based on the findings identifying extracellular vesicles (EVs) as intercellular conveyors of biological information. Complementary to traditional modes of signaling, EV-mediated signaling appears to be critical for metabolic cooperation and coordination between cells, tissues and organs. In this context, extra-cellular RNA (exRNA), which includes microRNAs, circular RNAs, and long non-coding RNA or lncRNAs, contained both within in EVs as well as in complex with lipoproteins, have been shown by us and others to regulate gene expression and alter cell function in various cell types. Moreover, during pathological conditions such as cancer, the number and compositions of EVs and exRNA change, altering the host immune response as well as synchronizing the behavior of secondary tumors. Isolations and analysis of EVs and exRNAs both during normal and pathological conditions are critical for understanding EVs and exRNAs biogenesis and their effector functions. This information is a critical prerequisite for novel disease diagnostic and prognostic strategies, biomarker-based surveillance for disease progression, treatment efficacy and relapse. An important determinant in EVs biogenesis is represented by the complement system, which upon activation generates membrane-targeting complexes, MACs (membrane attack complex, C5b-9), which represent ungated Ca++ and water channels. Following complement activation, targeted cells either exocytose and endocytose of the MAC-containing areas of the plasma membrane generating in the process large number of EVs. Currently, the study of EVs as biological entities relevant for intercellular signaling and disease diagnosis is based on the assumption that the biogenesis of EVs and exRNA happen at a steady state rate, being modified mostly by the healthy/diseased status of the host. Our preliminary data strongly suggest that that may not be the case. Our results show that the tissue-origin, number, protein and RNA composition, of EVs isolated by standard techniques depend not just on the blood collection methods, but also on the time of day the blood samples were collected. In this application, we propose to establish a baseline for the quality, quantity and composition of extracellular RNA, taking into account the potential artifacts induced by the: 1) blood collection methods, 2) time of collection, 3) physical activity. Our results obtained with fresh samples, will be cross-validated using stored plasma samples from the Center for Clinical Research and Division of Transfusion Medicine (NIH Blood Bank).