The Expression Levels of MicroRNA-361-5p and Its Target VEGFA Are Inversely Correlated in Human Cutaneous Squamous Cell Carcinoma Alexander Kanitz1, Jochen Imig1, Piotr J. Dziunycz2, Adriana Primorac1, Alessia Galgano1¤a, Gu¨ nther F. L. Hofbauer2, Andre´ P. Gerber1*¤b, Michael Detmar1* 1 Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland, 2 Department of Dermatology, University Hospital Zurich, Zurich, Switzerland Abstract Vascular endothelial growth factor A (VEGFA) plays a key role in the angiogenesis of human skin. Elevated levels of VEGFA are associated with several pathological conditions, including chronic inflammatory skin diseases and several types of skin cancer. In particular, squamous cell carcinoma (SCC) of the skin, the second most common skin cancer in the general population, is characterized by invasive growth, pronounced angiogenesis and elevated levels of VEGFA. The processing, turnover and production of VEGFA are extensively regulated at the post-transcriptional level, both by RNA-binding proteins and microRNAs (miRNAs). In the present study, we identified a new miRNA recognition element in a downstream conserved region of the VEGFA 39-UTR. We confirmed the repressive effect of miR-361-5p on this element in vitro, identifying the first target for this miRNA. Importantly, we found that miR-361-5p levels are inversely correlated with VEGFA expression in SCC and in healthy skin, indicating that miR-361-5p could play a role in cancers. Citation: Kanitz A, Imig J, Dziunycz PJ, Primorac A, Galgano A, et al. (2012) The Expression Levels of MicroRNA-361-5p and Its Target VEGFA Are Inversely Correlated in Human Cutaneous Squamous Cell Carcinoma. PLoS ONE 7(11): e49568. doi:10.1371/journal.pone.0049568 Editor: Amanda Ewart Toland, Ohio State University Medical Center, United States of America Received November 2, 2011; Accepted October 12, 2012; Published November 14, 2012 Copyright: 2012 Kanitz 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 supported by National Institutes of Health (http://www.nih.gov/) grant CA69184, Swiss National Science Foundation (http://www.snf.ch/) grants 3100A0_108207 and 31003A_130627, European Research Council (http://erc.europa.eu/) Advanced Grant LYVICAM (Lymphatic Vessels in Inflammation and Cancer Metastasis), Oncosuisse (http://www2.oncosuisse.ch/), Krebsliga Zurich (http://www.krebsliga-zh.ch/) (to MD), the Bonizzi-Theler Foundation, ETH Zurich (http://www.ethz.ch/) grant ETH-19 09-1, Swiss National Science Foundation grant CRSII3_127454 (to APG), EMDO foundation, Olga-Mayenfisch-Foundation (to GFLH), the European Skin Research Foundation ESRF (http://www.euroskinresearch.org/), and the Rene´ Touraine Foundation (http://www.fondation-r-touraine. org/) (to PJD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: Andre´ P. Gerber is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: a.gerber@surrey.ac.uk (APG); michael.detmar@pharma.ethz.ch (MD) ¤a Current address: Faculte´ de Me´decine, Universite´ Paris Descartes, Paris, France ¤b Current address: Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom Introduction Angiogenesis is a multi-step process leading to the formation of vascular structures derived from preexisting blood vessels, either through remodeling or the formation (‘‘sprouting’’) of new vessels. It involves the induction of microvascular hyperpermeability, breakdown of the vascular basement membrane, recruitment and proliferation of endothelial cells (EC), and the formation of mature blood vessels. Positive and negative regulators of angiogenesis have to be tightly regulated to maintain physiological tissue homeostasis and function. For example, in healthy adult skin, angiogenesis is generally quiescent as angiogenic stimuli are overruled by inhibitory signals. However, environmental insults may tip this balance, leading to initiation of angiogenesis in order to counteract tissue damage. Similarly, excessive angiogenesis in the skin, resulting from dysregulation of one or more of its regulators, is associated with a plethora of pathological conditions, such as psoriasis and other inflammatory dermatoses, autoimmune blis- tering diseases, and many cancers, most prominently melanoma, basal cell carcinoma and squamous cell carcinoma. Anti-angio- genic therapy therefore holds promise for the treatment of a wide spectrum of human ailments [1,2]. Cutaneous squamous cell carcinoma (SCC) is the second most common skin cancer in the general population [3]. In contrast to basal cell carcinoma – the most common skin cancer – it is characterized by the risk for metastasis. Incidence of SCC is 60- to 100-times higher among immunosuppressed patients, which makes it the most common cancer following organ transplantation. Invasive SCC develops from atypical keratinocytes, clinically visible as actinic keratosis or Bowen’s disease, both considered intraepithelial non-invasive forms of SCC [4]. Approximately 1% of these intraepithelial lesions develop into an invasive SCC [5]. However, such tumor development requires intense interactions with stromal cells and profound extracellular remodeling. Angio- genesis is an essential part of the malignant phenotype as most tumors are apparently not able to exceed 1–2 mm of diameter without developing new blood vessels [6]. Therefore they produce angiogenic factors at an early point of development. Vascular endothelial growth factor A (VEGFA) is a homo- dimeric heparin-binding glycoprotein that mainly acts as a para- crine mitogen, growth and survival factor for ECs, but it also causes vascular permeability, vasodilatation, and various changes in immune cell properties upon binding to its main receptors VEGF receptor 21 and 22. VEGFA has been identified as the PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49568