Detection of microRNA Expression in Human Peripheral Blood Microvesicles Melissa Piper Hunter1., Noura Ismail1,2., Xiaoli Zhang 3 , Baltazar D. Aguda4, Eun Joo Lee5, Lianbo Yu 3 , Tao Xiao6, Jeffrey Schafer1, Mei-Ling Ting Lee7, Thomas D. Schmittgen5, S. Patrick Nana-Sinkam1, David Jarjoura3, Clay B. Marsh1,2* 1 Division of Pulmonary, Allergy, Critical Care, Sleep Medicine, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America, 2 Molecular, Cellular, Developmental Biology Program, College of Biological Sciences, The Ohio State University, Columbus, Ohio, United States of America, 3 Center for Biostatistics, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America, 4 Mathematical Biosciences Institute, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America, 5 College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America, 6 Biostatistics, College of Public Health, The Ohio State University, Columbus, Ohio, United States of America, 7 Department of Epidemiology and Biostatistics, University of Maryland, College Park, Maryland, United States of America Abstract Background: MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis. Methodology/Principal Findings: Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real- time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function. Conclusions: This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA. Citation: Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, et al. (2008) Detection of microRNA Expression in Human Peripheral Blood Microvesicles. PLoS ONE 3(11): e3694. doi:10.1371/journal.pone.0003694 Editor: Yuk Ming Dennis Lo, Chinese University of Hong Kong, Hong Kong Received May 15, 2008; Accepted October 22, 2008; Published November 11, 2008 Copyright: 2008 Hunter et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was in part supported through funding from Battelle Memorial Institute and NHLBI grant (R01 HL067176). Dr. M. Piper Hunter is a Parker B. Francis Fellow in Pulmonary Research. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Clay.Marsh@osumc.edu . These authors contributed equally to this work. Introduction MicroRNAs (miRNAs) are small non-coding RNA sequences of about 22 nt [1]. miRNAs have been identified in animals, plants and viruses [2]. To date, advances in methodology have led to the identification of 587 human miRNAs (Sanger updated database [3]), and it is estimated that over 30% of human genes are targeted by these miRNAs [4]. miRNAs are implicated in fundamental cellular processes including hematopoietic differentiation [5,6], cell cycle regulation [7,8] and metabolism [9,10]. The evolutionary conserved miRNAs are transcribed by polymerase II and function as post- transcriptional gene regulators by targeting messenger RNA (mRNA) or proteins [11]. All precursor miRNA have stem loop structures, cleaved by Drosha and Dicer, to form the mature functional miRNA. Through formation of RNA-induced silencing complexes, miRNAs can either cleave mature mRNA molecules or inhibit their translation thus representing a newly discovered layer of gene regulation. MiRNAs play an important role in disease progression and carcinogenesis [12,13]; MiRNAs can act as oncogenes or tumor suppressors [14]. A study led by Calin et al. first revealed the connection between miR-15 and -16 and B-cell chronic lymphocytic leukemia [15]. Later, several studies showed the importance of miRNA in prognosis, initiation and progression of numerous cancer types [16–18]. Hence, utilizing miRNA fingerprinting as a novel diagnostic, prognostic and disease surveillance tool is of increasing importance [19]. PLoS ONE | www.plosone.org 1 November 2008 | Volume 3 | Issue 11 | e3694