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

描述（由申请人提供）： 摘要细胞外囊泡（EV）是细胞间生物信息的传递者，这一发现为旁分泌信号转导提供了新的范式。作为传统信号传导模式的补充，EV介导的信号传导似乎对细胞、组织和器官之间的代谢合作和协调至关重要。在这种情况下，细胞外RNA（exRNA），包括微小RNA、环状RNA和长的非编码RNA或lncRNA，包含在EV内以及与脂蛋白复合，已经被我们和其他人证明在各种细胞类型中调节基因表达并改变细胞功能。此外，在癌症等病理条件下，EV和exRNA的数量和组成发生变化，改变宿主免疫应答以及同步继发性肿瘤的行为。在正常和病理条件下EV和exRNA的分离和分析对于理解EV和exRNA生物发生及其效应子功能至关重要。这些信息是新的疾病诊断和预后策略、基于生物标志物的疾病进展监测、治疗疗效和复发的关键先决条件。EV生物发生中的一个重要决定因素是补体系统，其在激活时产生膜靶向复合物，MAC（膜攻击复合物，C5 b-9），其代表非门控Ca++和水通道。在补体激活后，靶细胞外吞和内吞质膜的含MAC区域，在此过程中产生大量EV。目前，EV作为与细胞间信号传导和疾病诊断相关的生物实体的研究是基于以下假设：EV和exRNA的生物发生以稳态速率发生，主要由宿主的健康/患病状态改变。我们的初步数据强烈表明，情况可能并非如此。我们的研究结果表明，通过标准技术分离的EV的组织来源、数量、蛋白质和RNA组成不仅取决于血液采集方法，还取决于血液样本采集的时间。在本申请中，我们建议建立细胞外RNA的质量、数量和组成的基线，同时考虑到由以下因素引起的潜在伪影：1）血液采集方法，2）采集时间，3）体力活动。我们用新鲜样品获得的结果，将 使用临床研究中心和输血医学部（NIH血库）储存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).