New Target Genes of MITF-Induced microRNA-211 Contribute to Melanoma Cell Invasion Christiane Margue . , Demetra Philippidou . , Susanne E. Reinsbach, Martina Schmitt, Iris Behrmann, Stephanie Kreis* Signal Transduction Laboratory, Life Sciences Research Unit, University of Luxembourg, Luxembourg, Luxembourg Abstract The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF). By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR- 211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma. Citation: Margue C, Philippidou D, Reinsbach SE, Schmitt M, Behrmann I, et al. (2013) New Target Genes of MITF-Induced microRNA-211 Contribute to Melanoma Cell Invasion. PLoS ONE 8(9): e73473. doi:10.1371/journal.pone.0073473 Editor: Soheil S. Dadras, University of Connecticut Health Center, United States of America Received March 28, 2013; Accepted July 19, 2013; Published September 5, 2013 Copyright: ß 2013 Margue 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 study was funded by a research grant from the University of Luxembourg (F1R_LSC-PUL-09MIRN). 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 declared that no competing interests exist. * E-mail: stephanie.kreis@uni.lu . These authors contributed equally to this work. Introduction Most likely owing to changed sun-tanning behavior, cutaneous melanoma is rapidly increasing in the industrialized world: an estimated 76,250 people will be diagnosed with invasive melano- ma in 2012 in the US, where the incidence increased by 45% from 1992 to 2004 (www.skincancer.org). To this day, molecular markers that would permit differential diagnosis at early stages, classification and prediction of disease progression that could help clinicians to offer more personalized and effective treatment options are not yet consolidated. In this context, miRNAs have been extensively studied to become the sought-after biomarkers, clinical targets, or predictors of cancer and other diseases [1–3]. Profiling of miRNA levels and investigation of functional consequences of individual or groups of miRNAs is complicated by their relatively small variations in expression levels (between 1.5–5 fold) [4]. Nevertheless, even small differences in miRNA expression levels might have profound functional consequences for cellular growth, proliferation, differentiation, and other funda- mental cellular programs [5]. Another striking denominator of miRNA profiling in many cancers, including melanoma, is the heterogeneity of their basal miRNA levels. The limitations of such profiling studies have recently been reviewed [6] and can be attributed to factors such as technical issues, intrinsic cellular heterogeneity of the clinical samples, different sample types, and the original driving event for development of melanoma (genetic, UV radiation, etc.). Therefore, it remains difficult to this day to summarize existing data in one reliable melanoma-specific miRNA expression set. Nonetheless, one of few miRNAs that seem specific to the melanocyte lineage is miR-211 [7,8]. Most studies concur on a down-regulation or a complete loss of this miRNA in the majority of analyzed melanoma patient samples and cell lines when compared to normal skin, nevi or NHEM (normal human melanocytes) [9–21]. On the other hand, Mueller et al. did not detect a differential expression of miR-211 in melanoma cell lines versus normal melanocytes [22] and even elevated miR-211 levels in melanoma samples relative to nevi have been described [23]; high expression levels of miR-211 have further been used to discriminate between melanoma and other cancer cell lines [7,8]. MITF (microphthalmia-associated transcription factor), the master regulator of melanocyte proliferation, survival and differentiation, has intricate regulatory roles in melanoma development [24]. Described as a ‘‘lineage addiction’’ oncogene, MITF is amplified in 10–20% of melanomas; however, MITF has also been attributed tumor-suppressive roles [25]. One of the PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e73473