Plasma microRNA Profile in Canine Appendicular Osteosarcoma Patients by Michael Edson A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Pathobiology Guelph, Ontario, Canada © Michael Edson, September, 2019 ABSTRACT PLASMA MICRORNA PROFILE IN CANINE APPENDICULAR OSTEOSARCOMA PATIENTS Michael Edson Advisor: University of Guelph, 2019 Dr. Geoffrey Wood Osteosarcoma (OSA) is the most common primary bone tumor in dogs. The standard of care for canine appendicular OSA involves amputation followed by chemotherapy. Currently, there are no established biomarkers to determine which dogs benefit the most from chemotherapy. MicroRNAs (miRNAs) are small non-coding RNAs that are potential biomarkers of osteosarcoma. Plasma miRNA levels were profiled in healthy dogs and OSA patients, collected before and after amputation. Quantitative real-time PCR was conducted to determine the levels of 277 miRNAs on a miRNome PCR array. From these findings, a custom PCR array was designed and used to examine miRNA levels and correlations to clinical outcome in 20 control and 28 OSA cases. Additionally, miRNA levels in erythrocytes were analyzed. Overall, 37 plasma miRNAs and 1 erythrocyte miRNA had significant level changes. Furthermore, 24 plasma miRNAs were correlated to clinical outcome. These miRNAs could improve decision making in treatments for OSA patients. iii ACKNOWLEDGEMENTS I’d like to thank my advisor, Dr. Geoffrey Wood, as well as my committee members, Dr. Anthony Mutsaers, Dr. Alicia Viloria-Petit, and Dr. R. Darren Wood for their advice and guidance on this project. Many thanks to Mary Ellen Clark, Dr. Karlee Craig, Gabhan Chalmers and Leah Read for their technical assistance and advice in the laboratory. Special thanks to Drs. Karolina Skowronski and Vicki Sabine of the Institute for Comparative Cancer Investigation for their help with collecting plasma and erythrocyte samples as well as patient data. Thanks also to Sylvie Becker for her assistance with plasma RNA extractions, Meagan Parmassar for her work on the erythrocyte hemolysis project and Jessica Woodman for her efforts on miRNA sequence comparisons. Finally, thanks to the clients and patients who kindly provided plasma and erythrocyte samples. Financial assistance was graciously provided by the OVC Pet Trust, OVC MSc Scholarship and the University of Guelph Graduate Entrance Excellence Scholarship. iv TABLE OF CONTENTS ABSTRACT.............................................................................................................................. ii ACKNOWLEDGEMENTS .................................................................................................... iii TABLE OF CONTENTS ........................................................................................................ iv LIST OF TABLES ................................................................................................................. vii LIST OF FIGURES ................................................................................................................ ix LIST OF ABBREVIATIONS ................................................................................................. xi LIST OF APPENDICES ....................................................................................................... xiii CHAPTER 1: Literature Review..............................................................................................1 1.1 INTRODUCTION ............................................................................................................................................. 1 1.2 CANINE OSTEOSARCOMA ............................................................................................................................... 2 1.2.1 Diagnosis .............................................................................................................................................. 2 1.2.2 Classification ........................................................................................................................................ 3 1.2.3 Risk Factors .......................................................................................................................................... 6 1.2.4 Treatment .............................................................................................................................................. 8 1.2.5 Canine Osteosarcoma as a Model for Human Osteosarcoma.................................................................. 9 1.3 BIOMARKERS .............................................................................................................................................. 10 1.4 MICRORNAS .............................................................................................................................................. 13 1.4.1 Biosynthesis ........................................................................................................................................ 14 1.4.2 Nomenclature ...................................................................................................................................... 15 1.4.3 Clusters ............................................................................................................................................... 15 1.4.4 Stability ............................................................................................................................................... 16 1.4.5 Hemolysis ............................................................................................................................................ 18 1.4.6 Conservation between Species ............................................................................................................. 21 1.4.7 Dysregulation in Cancer ...................................................................................................................... 22 1.4.8 MicroRNAs in Canine OSA .................................................................................................................. 24 1.4.9 Summary ............................................................................................................................................. 27 CHAPTER 2: Preliminary Study ........................................................................................... 29 2.1 HYPOTHESES ............................................................................................................................................... 29 2.2 MATERIALS AND METHODS ......................................................................................................................... 29 2.2.1 Sample Collection................................................................................................................................ 29 2.2.2 RNA Extraction ................................................................................................................................... 32 2.2.3 Reverse-Transcription PCR ................................................................................................................. 33 2.2.4 Quantitative Real-time PCR ................................................................................................................. 33 2.2.5 Data Analysis ...................................................................................................................................... 34 2.3 RESULTS ..................................................................................................................................................... 35 2.3.1 Patient Demographics ......................................................................................................................... 35 v 2.3.2 Normalization...................................................................................................................................... 35 2.3.3 Hemolysis Detection ............................................................................................................................ 36 2.3.4 miRNA Expression ............................................................................................................................... 36 2.3.5 miRNAs from Literature....................................................................................................................... 39 2.3.6 Control miRNAs .................................................................................................................................. 40 2.3.7 Custom PCR Array Design .................................................................................................................. 41 2.4 DISCUSSION ................................................................................................................................................ 42 CHAPTER 3: Focused Study ................................................................................................. 50 3.1 HYPOTHESES/OBJECTIVES ........................................................................................................................... 50 3.2 MATERIALS AND METHODS ......................................................................................................................... 50 3.2.1 Sample Collection................................................................................................................................ 50 3.2.2 RNA Extraction ..................................................................................................................................