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Mir-34A Regulates the Invasive Capacity of Canine Osteosarcoma Cell Lines

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Mir-34A Regulates the Invasive Capacity of Canine Osteosarcoma Cell Lines MiR-34a Regulates the Invasive Capacity of Canine Osteosarcoma Cell Lines THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Cecilia Montes Lopez Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2017 Master's Examination Committee: Joelle Fenger, DVM, PhD, DACVIM, Advisor Emma Warry, BVSc (HONS), DACVIM William Kisseberth, DVM, PhD, DACVIM Copyrighted by Cecilia Montes Lopez 2017 Abstract Osteosarcoma (OSA) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a fundamental role in cancer. The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well- established spontaneous model of the human disease. Real-time PCR demonstrated that miR-34a expression levels were significantly reduced in primary canine OSA tumors and canine OSA cell lines as compared to normal canine osteoblasts. In canine OSA cell lines stably transduced with empty vector or pre- miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant down-regulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of down-regulated miR- 34a target genes showed enrichment of several biological processes related to cell invasion and motility. Lastly, we validated changes in miR-34a target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OSA cells ii overexpressing miR-34a. Concordant with these data, primary canine OSA tumor tissues demonstrated increased expression levels of putative miR-34a target genes. These data demonstrate that miR-34a contributes to invasion and migration in canine OSA cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA. iii Dedicated to Aaron Lopez for his incredible support, love, and companionship throughout this journey; to Veronica, Kevin, and Kathleen Montes for their love throughout my life; and in loving memory of Papi, siempre. iv Acknowledgments I would like to sincerely thank my incredible mentor, Dr. Joelle Fenger, for her patient guidance and teaching ability in this project. Thank you for making me excited about science! My gratitude goes out to the veterinary oncology faculty at The Ohio State University, including Drs. Cheryl London, William Kisseberth, Emma Warry, Megan Brown, Eric Green, Noopur Desai, and Vincent Wavreille who have shared their experience and abilities and helped to shape me professionally throughout my residency training. These individuals have helped to make these three years some of the most formative, educational, and progressive of my life. The contribution of the following collaborators was indispensable to this work: Peter Wu for his assistance with miR-34a target identification, and Xiaoli Zhang and Ayse Selen Yilmaz for their efforts with statistical analysis. I would also like to express my gratitude to Tim Vojt of the OSU College of Veterinary Medicine Biomedical Media Services for his assistance in figure preparation, Feng Xu for her technical assistance, and the Veterinary Clinical Research Shared Resource Biospecimen Repository for their assistance in tumor sample acquisition. I would like to thank my past and present residentmates, Pachi Clemente, Megan Brown, Heather Gardner, Rachel Kovac, and Kyle Renaldo, for their encouragement, friendship, and humor. And importantly I would like to thank the veterinary technicians v of the Integrated Oncology Service, Heather Young, Krista Cebulskie, Nicole Pastorek, Matt Kerzie, and Erin Mash, for their good-natured support. vi Vita 2006................................................................Lake Dallas High School 2010................................................................B.S. Biomedical Sciences, Texas A&M University 2013................................................................D.V.M, Texas A&M University 2013-2014 .....................................................Rotating Small Animal Intern, VCA Veterinary Specialty Center of Seattle 2014 to present ...............................................Combined Medical Oncology Residency and Masters Program, The Ohio State University Fields of Study Major Field: Comparative and Veterinary Medicine vii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii Table of Contents ............................................................................................................. viii List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii Chapter 1: Literature Review ............................................................................................. 1 Chapter 1.1 Functions of MicroRNAs in Health and Disease ....................................... 1 Chapter 1.2 MicroRNA Dysregulation in Cancer ........................................................... 2 Chapter 1.3 The miR-34 Family of MicroRNAs in Cancer ........................................... 4 Chapter 1.4 MiR-34 Dysregulation in Human Osteosarcoma ....................................... 6 Chapter 1.5 Comparative Aspects of Human and Canine Osteosarcoma ....................... 8 Chapter 2: MiR-34a Regulates the Invasive Capacity of Canine Osteosarcoma Cell Lines ........................................................................................................................................... 11 2.1 Abstract ................................................................................................................... 11 viii 2.2 Introduction ............................................................................................................. 12 2.3 Materials and Methods ............................................................................................ 15 2.3.1 Cell lines and primary tumor samples .............................................................. 15 2.3.2 RNA isolation, cDNA synthesis, and quantitative real-time PCR ................... 16 2.3.3 MiR-34a lentivirus infection ............................................................................ 17 2.3.4 Assessment of cell proliferation ....................................................................... 18 2.3.5 Cell-cycle analysis ............................................................................................ 18 2.3.6 Matrigel invasion assay .................................................................................... 19 2.3.7 Wound healing assay ........................................................................................ 19 2.3.8 RNA sequencing ............................................................................................... 20 2.3.9 Statistics ............................................................................................................ 21 2.4 Results ..................................................................................................................... 21 2.4.1 MiR-34a expression is decreased in primary canine OSA tumors and canine OSA cell lines ............................................................................................................ 21 2.4.2 MiR-34a expression is decreased in paired canine OSA metastatic lesions compared to primary OSA tumors ............................................................................. 22 2.4.3 Ectopic expression of miR-34a does not influence cell proliferation or cell cycle distribution in canine OSA cell lines ............................................................... 22 2.4.5 RNA sequencing identifies miR-34a-induced gene alterations in canine OSA8 cells ............................................................................................................................ 24 ix 2.4.6 Putative miR-34a target gene expression is increased in primary canine OSA tumors ........................................................................................................................ 25 2.5 Discussion ............................................................................................................... 26 2.6 Future Directions ..................................................................................................... 31 2.7 Conclusions ............................................................................................................. 33 References ......................................................................................................................... 46 Appendix
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