Abstract 184: Breed-associated differential microRNA expression in canine osteosarcoma

Abstract

Abstract Introduction: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. miRNAs are dysregulated in cancer, suggesting they play a role in tumorigenesis. Osteosarcoma (OSA) is the most common bone tumor in dogs, however, little is known regarding mechanisms underlying malignant transformation in these tumors. Breeds such as Rottweilers and Greyhounds are at higher risk for developing OSA, suggesting that heritable factors play a role in this disease. We hypothesize that dysregulation of miRNAs in canine OSA is associated with specific breeds. Methods: RNA was isolated by the Trizol (Invitrogen) method from a panel of seven normal canine tissues and 48 primary canine OSA tumors from Greyhound, Golden Retriever, Rottweiler, and mixed breed dogs. miRNA expression was analyzed using the NanoString nCounter human microRNA Expression Assay, interrogating the expression of 752 human miRNAs; 168 of whose mature sequences are 100% conserved between human and dog (Sanger miRBase V15). Samples were hybridized to reporter CodeSets, processed on the nCounter prep station, and scanned with the nCounter Digital Analyzer. Nanominer software was used to perform data normalization. Real time PCR was performed using Applied Biosystems Taqman miRNA assays. Normalization was performed with U6 snRNA and miRNA expression was calculated utilizing the comparative Ct method. P-values of <0.05 were considered statistically significant. For reverse-transcription PCR reactions, cDNA was prepared using Superscript III (Invitrogen) and PCR was performed using ThermoPrime Taq Polymerase (ThermoScientific). Results: miRNA profiling of normal canine tissues revealed tissue-specific miRNA expression signatures. Real time PCR validation of tissue-specific miRNAs validated the use of the NanoString nCounter Assay as a platform for evaluating miRNA expression in canine tissues. Supervised hierarchical cluster analysis revealed distinct breed-associated miRNA expression signatures in canine OSA. 189 miRNAs were differentially expressed in Greyhound, Rottweiler, Golden Retriever, and mixed breed tumors (p<0.01). In an expanded cohort of Greyhound and Rottweiler tumors, real time PCR demonstrated that miR-494 is highly expressed in Rottweiler OSA as compared to Greyhound OSA (p<0.05) or normal canine osteoblasts from various dog breeds, including one Rottweiler. Studies are underway to assess the biological consequences of miR-494 overexpression in normal canine osteoblasts and OSA cell lines. Small RNA sequencing of these OSA samples using the Applied Biosystems SOLiD 4 sequencing platform is being performed to further define breed-associated miRNA signatures in canine OSA. Conclusions: These data reveal significant correlations between breed and miRNA expression in canine OSA, suggesting breed-associated patterns of miRNA dysregulation may play a role in the pathogenesis of OSA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 184. doi:1538-7445.AM2012-184