Genome-Wide Profiling of MicroRNAs in Adipose Mesenchymal Stem Cell Differentiation and Mouse Models of Obesity Lena Bengestrate1¤, Sam Virtue2, Mark Campbell2, Antonio Vidal-Puig2, Dirk Hadaschik1, Peter Hahn 1 , Wolfgang Bielke1* 1 Department of Epigenetics, QIAGEN GmbH, Hilden, Germany, 2 Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom Abstract In recent years, there has been accumulating evidence that microRNAs are key regulator molecules of gene expression. The cellular processes that are regulated by microRNAs include e.g. cell proliferation, programmed cell death and cell differentiation. Adipocyte differentiation is a highly regulated cellular process for which several important regulating factors have been discovered, but still not all are known to fully understand the underlying mechanisms. In the present study, we analyzed the expression of 597 microRNAs during the differentiation of mouse mesenchymal stem cells into terminally differentiated adipocytes by real-time RT-PCR. In total, 66 miRNAs were differentially expressed in mesenchymal stem cell- derived adipocytes compared to the undifferentiated progenitor cells. To further study the regulation of these 66 miRNAs in white adipose tissue in vivo and their dependence on PPARc activity, mouse models of genetically or diet induced obesity as well as a mouse line expressing a dominant negative PPARc mutant were employed. Citation: Bengestrate L, Virtue S, Campbell M, Vidal-Puig A, Hadaschik D, et al. (2011) Genome-Wide Profiling of MicroRNAs in Adipose Mesenchymal Stem Cell Differentiation and Mouse Models of Obesity. PLoS ONE 6(6): e21305. doi:10.1371/journal.pone.0021305 Editor: Alfons Navarro, University of Barcelona, Spain Received March 22, 2011; Accepted May 25, 2011; Published June 23, 2011 Copyright: 2011 Bengestrate 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: The work for this project was funded in part by MRC Programme grant number 14133 (www.mrc.ac.uk/index.htm) and BBSRC grant number 20485 (www.bbsrc.ac.uk) to Antonio Vidal-Puig. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors DH, PH, and WB are currently employees within the R&D department of QIAGEN GmbH and declare potential competing financial interests, including personal ownership of QIAGEN stocks and/or stock options. Several commercially available products, marketed by QIAGEN, were employed for this study. These products include kits and items for nucleic acid isolation and amplification, as well as the miScript system for microRNA detection. QIAGEN holds several issued and pending patents related to these products. The above said does not alter the authors’ adherence to all the PloS ONE policies on sharing data and materials. * E-mail: wolfgang.bielke@qiagen.com ¤ Current address: Institute for Biochemistry II, University of Cologne, Cologne, Germany Introduction During the last decades, obesity and associated diseases, such as type 2 diabetes, hypertension, and cardiovascular disease, have reached an epidemiologic proportion [1,2]. This fact has led to intensive studies, conducted with the help of suitable cell culture systems, supplemented by transgenic- or knock-out mouse models to investigate the genetic or environmental factors influencing obesity. The employment of such mouse models has revealed more than 200 genes involved in the regulation of fat metabolism and adiposity. These genes may affect the regulation of food uptake, differentiation of preadipocytes, or fat storage capability of adipocytes [3]. Although the role of many protein-encoding genes has been well established, much less is known about the regulatory influence of microRNAs (miRNAs) on proteins or gene-transcripts, involved in these processes. microRNAs are defined as a class of short interfering RNAs, which are capable to bind to partially complementary sequences localized within the 39-untranslated regions (39-UTRs) of mRNAs. miRNAs affect gene expression as part of a multi-modal protein complex, called RNA Induced Silencing Complex (RISC). Binding of miRNAs to target mRNAs results in translational repression or degradation of the targeted transcript. Cumulating evidence allocates miRNA functions to integral parts of regulatory cellular processes, including the differentiation of stem cell populations, the regulation of metabolic activities, and tumorigenesis [4,5,6]. Multipotent adult mesenchymal stem cells (MSC) are capable of differentiating into a variety of cell types, including adipocytes, osteoblasts, chondrocytes, and myocytes [7,8,9]. Regulation of stemcellness vs. differentiation is a key event in mesenchymal stem cell biology, and allows appropriate tissue homeostasis, while maintaining a sufficient pool of precursor cells for further use. Such pools of mesenchymal stem cells, residing in the bone marrow or the vascular stroma of adipose tissue, are also the source for MSCs, which ultimately develop into adult adipocytes [10,11,12]. In vitro, MSCs proliferate and develop into preadipo- cytes after reaching confluency. Preadipocytes are still competent for proliferation but, when allowed to become confluent and treated with substances such as insulin and glucocorticoids, cease cell division and differentiate into adipocytes, [13]. At the molecular level, the core process of adipogenesis is regulated by transcription factors, such as CCAAT/enhancer binding protein a (C/EBP-a) and peroxisome proliferator activated receptor c (PPARc), a heterodimerization partner of Retinoic X receptors PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21305