Breed Associated miRNA Expression in Canine Osteosarcoma

Professional Biological Sciences: 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)Introduction: MicroRNAs (miRNAs) are small non-protein coding RNAs that have been implicated in humans as having a fundamental role in cancer initiation and progression. Osteosarcoma (OSA) is the most common bone tumor in dogs, however, little is known regarding mechanisms underlying malignant transformation in these tumors. Certain 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. In this study, we sought to characterize the expression of miRNAs in canine primary OSA tumors among Grehyounds, Rottweilers, Golden Retrievers, a mixed population of other breed and determine the association of differentially expressed miRNAs in primary tumors with breed. Methodology: Total RNA was isolated by the Trizol (Invitrogen) method from a panel of 7 normal canine tissues and 48 primary canine OSA tumors from Greyhound, Golden Retriever, Rottweiler, and mixed breed dogs. Mature miRNA expression in canine tissues was analyzed using the NanoString nCounter human microRNA Expression Assay, interrogating the expression profile of 752 human miRNAs; 168 of whose mature sequences are 100% conserved between human and dog (Sanger miRBase V15). 100 ng RNA per sample was hybridized overnight to the microRNA Expression Assay CodeSet and hybridized reactions were loaded onto the nCounter prep station for subsequent binding and washing steps. Cartridge scanning and quantification was performed with the nCounter Digital Analyzer. Nanominer software was used to perform raw data count normalization and p-values of <0.05 were considered statistically significant. To determine miR-494 expression levels in available canine osteosarcoma and osteoblast cell lines, RNA was isolated from 8 canine osteosarcoma cell lines, a commercially available canine osteoblast cell line (CellApp, Inc), and primary canine bone-derived cells obtained from 5 independent dogs (including Greyhound, Rottweiler, Golden Retriever, and mixed breed dogs). Real time PCR to detect mature miRNA expression was performed using Taqman miRNA assays on the Applied Biosystems StepOne Plus Detection System. Normalization was performed with the small nuclear RNA U6 and miRNA expression was calculated utilizing the comparative Ct method. Statistical analysis was performed using two-tailed paired Student’s t test for qRT-PCR and p-values of <0.05 were considered statistically significant. For primary canine bone-derived cells, reverse-transcription PCR to detect the expression of bone markers ALP, OP, BMP2 was performed using Superscript III (Invitrogen) and ThermoScientific ThermoPrime Taq Polymerase. To assess the effects of miR-494 overexpression on osteosarcoma cell behavior, canine osteosarcoma OSA16 cells (established from a Rottweiler dog) and commercially available normal canine osteoblasts were transduced with either the lentiviral pre-miR-494-GFP vector (System Biosciences) or lentiviral pCDH-CMVMCS-EF1-copGFP empty control vector (System Biosciences) and sorted using the iCyte Reflection high speed cell sorter. MiR-494 expression was performed as described above. Results and Conclusions: miRNA profiling of a panel of normal canine tissues revealed tissue-specific miRNA expression signatures. Independent real time PCR validation of a subset 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 one of these, miR-494 (a miRNA with known importance in human tumorigenesis) is highly expressed in primary Rottweiler OSA as compared to Greyhound OSA (p