title: MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines.
abstract: MicroRNAs (miRNAs) regulate the expression of networks of genes and their dysregulation is well documented in human malignancies; however, limited information exists regarding the impact of miRNAs on the development and progression of osteosarcoma (OS). Canine OS exhibits clinical and molecular features that closely resemble the corresponding human disease and it is considered a well-established spontaneous animal model to study OS biology. The purpose of this study was to investigate miRNA dysregulation in canine OS.
  
  
  
  We evaluated miRNA expression in primary canine OS tumors and normal canine osteoblast cells using the nanoString nCounter system. Quantitative PCR was used to validate the nanoString findings and to assess miR-9 expression in canine OS tumors, OS cell lines, and normal osteoblasts. Canine osteoblasts and OS cell lines were stably transduced with pre-miR-9 or anti-miR-9 lentiviral constructs to determine the consequences of miR-9 on cell proliferation, apoptosis, invasion and migration. Proteomic and gene expression profiling of normal canine osteoblasts with enforced miR-9 expression was performed using 2D-DIGE/tandem mass spectrometry and RNA sequencing and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. OS cell lines were transduced with gelsolin (GSN) shRNAs to investigate the impact of GSN knockdown on OS cell invasion.
  
  
  
  We identified a unique miRNA signature associated with primary canine OS and identified miR-9 as being significantly overexpressed in canine OS tumors and cell lines compared to normal osteoblasts. Additionally, high miR-9 expression was demonstrated in tumor-specific tissue obtained from primary OS tumors. In normal osteoblasts and OS cell lines transduced with miR-9 lentivirus, enhanced invasion and migration were observed, but miR-9 did not affect cell proliferation or apoptosis. Proteomic and transcriptional profiling of normal canine osteoblasts overexpressing miR-9 identified alterations in numerous genes, including upregulation of GSN, an actin filament-severing protein involved in cytoskeletal remodeling. Lastly, stable downregulation of miR-9 in OS cell lines reduced GSN expression with a concomitant decrease in cell invasion and migration; concordantly, cells transduced with GSN shRNA demonstrated decreased invasive properties.
  
  
  
  Our findings demonstrate that miR-9 promotes a metastatic phenotype in normal canine osteoblasts and malignant OS cell lines, and that this is mediated in part by enhanced GSN expression. As such, miR-9 represents a novel target for therapeutic intervention in OS.
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, 601 Vernon L. Tharp Street, Columbus, OH, USA. fenger.3@osu.edu.|, 444 Veterinary Medical Academic Building, 1600 Coffey Road, Columbus, OH, 43210, USA. fenger.3@osu.edu.
authors.forename: Joelle M
authors.initials: JM
authors.lastname: Fenger
authors.affiliation: Center for Childhood Cancer, Nationwide Children's Hospital, 700 Children's Drive, Columbus, OH, USA.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Roberts
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, 129 Hamilton Hall, 1645 Neil Avenue, Columbus, OH, USA.
authors.forename: O Hans
authors.initials: OH
authors.lastname: Iwenofu
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.
authors.forename: Misty D
authors.initials: MD
authors.lastname: Bear
authors.affiliation: Center for Biostatistics, The Ohio State University, 320B Lincoln Tower, 1800 Cannon Drive, Columbus, OH, USA.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, 601 Vernon L. Tharp Street, Columbus, OH, USA.
authors.forename: Jason I
authors.initials: JI
authors.lastname: Couto
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN, USA.|Masonic Cancer Center, University of Minnesota, 420 Delaware Street, SE, MMC 806, Minneapolis, MN, USA.
authors.forename: Jaime F
authors.initials: JF
authors.lastname: Modiano
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, 601 Vernon L. Tharp Street, Columbus, OH, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Kisseberth
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, 601 Vernon L. Tharp Street, Columbus, OH, USA.|Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: London
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001859:Bone Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D016000:Cluster Analysis; D004195:Disease Models, Animal; D004285:Dogs; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D035683:MicroRNAs / Q000235:genetics*; D009362:Neoplasm Metastasis; D010006:Osteoblasts / Q000378:metabolism; D012516:Osteosarcoma / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology*; D010641:Phenotype; D040901:Proteomics / Q000379:methods; D059467:Transcriptome
chemical_list: D035683:MicroRNAs; D020169:Caspases
keywords: Canine; Comparative oncology; MicroRNA; Osteosarcoma; miR-9