miR-514a regulates the tumour suppressor NF1 and modulates BRAFi sensitivity in melanoma

Abstract

// Mitchell S. Stark 1,2 , Vanessa F. Bonazzi 3 , Glen M. Boyle 1 , Jane M. Palmer 1 , Judith Symmons 1 , Catherine M. Lanagan 1 , Christopher W. Schmidt 1 , Adrian C. Herington 2 , Robert Ballotti 3 , Pamela M. Pollock 2 and Nicholas K. Hayward 1 1 QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD, Australia 2 School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia 3 Inserm U1065, Centre Méditerranéen de Médecine Moléculaire, Equipe 1, Biologie et pathologies des mélanocytes, Nice, France Correspondence to: Mitchell S. Stark, email: // Keywords : miRNA, microRNA, miR-514a, BRAFi, NF1 Received : March 16, 2015 Accepted : April 07, 2015 Published : April 23, 2015 Abstract To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1 , CDK2 , MC1R , and NF1 , previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E -targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies. Mitchell S. Stark