Proceeding of Veterinary and Animal Science Days 2017, 6th- 8th June, Milan, Italy HAF © 2013 Vol. IV, No. 1s ISSN: 2283-3927 l Keywords Canine oral melanoma, Canine cutaneous melanoma, MiRNA, FFPE sample CORRESPONDING AUTHOR Carlotta Catozzi carlotta.catozzi@unimi.it JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Expression Analysis of MicroRNAs in FFPE Samples of canine cutaneous and oral melanoma by RT-qPCR Carlotta Catozzi1*, Cristina Lecchi1, Chiara Gini1, Fabrizio Ceciliani1, Riccardo Finotello2, Lorenzo Ressel3 1University of Milan, Department of Veterinary Medicine, Italy 2University of Liverpool, Department of Small Animal Clinical Science, Neston, United Kingdom 3University of Liverpool, Department of Veterinary Pathology and Public Health, Neston, United Kingdom Abstract MicroRNA (miRNA), a class of small, non-coding RNA - regulating post-transcriptionally protein expression - are emerging as clinical biomarkers in many pathologies, including cancer (Peng and Croce, 2016). Since miRNA are supposed to represent fundamental key regulators, better understanding of melanoma tumour biology is essential to improve both disease grading and staging and, consequently, therapy options and prognosis. The aim of the study was to investigate whether miRNA expression can vary in canine melanoma samples derived from formalin-fixed-paraffin- embedded (FFPE) tissues. Experimental design of the study included three groups, each one composed of 7 animals: i) control healthy skin group ii) oral melanoma group iii) skin melanoma group. Two tissue slides were used for miRNA extraction. The expression levels of seven miRNA - miR-145, miR-146a, miR-425-5p, miR-223, miR-365, miR-155 and miR-134 - were detected and assessed by qPCR using TaqMan® probes (Veramo et al, 2017; Segura et al., 2012; Wagner et al., 2013; Sand et al.,2013). Five miRNA were significantly up-(n=3) or down-(n=2) regulated. In details, miR-146a and miR-155 abundance was increased as compared with control in both oral and skin melanoma (Fig 1 B,E) (p = 0.004 and 0.014 and p = 0.043 and 0.035 respectively), while the levels of miR-145 and miR-365 were lower (Fig 1 A,D) (p = 0.018 and 0.008 and p = 0.01 and 0.028, respectively). MiR-425-5p was upregulated (p = 0.039) only in skin melanoma (Fig. 1 C). Furthermore, functional analysis, carried out using miRNet web-based tool, showed that 76 genes related to cancer-associated pathways were possible target of these five microRNA (p = 6.95E-9); in particular, 21 target genes were associated with melanoma (p = 1.47E-5), including BRAF and CDK, E2F, FGF and PIK3 families. In conclusion, miR- 145, miR-146a, miR-425-5p, miR-365 and miR-155 are differentially expressed in melanoma and healthy FFPE samples, suggesting that they may play a role in canine melanoma pathogenesis and/or progression. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en Proceeding of Veterinary and Animal Science Days 2017, 6th- 8th June, Milan, Italy HAF © 2013 Vol. IV, No. 1s ISSN: 2283-3927 Fig 1: Boxplots of the median (black line), the mean (+), 25%- and 75%-quartile and range of the values measured, as well as statistical differences between groups are shown for miR-145, miR-146a, miR-425-5p, miR-365 and miR-155 in control, oral melanoma (oral M) and cutaneous melanoma (skin M) samples References Peng and Croce, 2016. The role of MicroRNAs in human cancer. Signal Transduction and Targeted Therapy. 1, 15004 Varamo et al., 2017. MicroRNAa role as potential biomarkers and key regulators in Melanoma. Genes, chromosomes and cancer. 56, 3-10 Segura et al., 2012. MicroRNA and cutaneous melanoma: from discovery to prognosis and therapy. Carcinogenesis. 33, 1823–1832 Wagner et al., 2013. Comparison of non-coding RNAs in human and canine cancer. Frontiers in Genetics. 4, 46-2 Sand et al., 2013. Comparative microarray analysis of microRNA expression profiles in primary cutaneous malignant melanoma, cutaneous malignant melanoma metastases, and benign melanocytic nevi. Cell Tissue Res. 351(1), 85-98 http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en