The Role of MicroRNA-140 and its IsomiRs in an In Vitro model of Osteoarthritis Doctoral thesis by Rua Nader Al-Modawi Institutes of Clinical Medicine and Basic Medical Science Departments of Immunology and Orthopedics and Norwegian Center for Stem Cell Research Oslo University Hospital UNIVERSITY OF OSLO Faculty of Medicine 2020 © Rua Nader Al-Modawi, 2021 Series of dissertations submitted to the Faculty of Medicine, University of Oslo ISBN 978-82-8377-823-6 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo. Humility is an indispensable companion in any endeavor pursuing the truth. Table of Contents Acknowledgment ........................................................................................................ 1 Abbreviations .............................................................................................................. 3 Publications included .................................................................................................. 6 1 Introduction ............................................................................................................. 7 1.1 Cartilage ..................................................................................................... 7 1.1.1 Articular Cartilage .................................................................................. 7 1.2 Cartilage injuries and their treatment ...................................................... 10 1.3 Osteoarthritis ........................................................................................... 13 1.3.1 Epidemiology ....................................................................................... 13 1.3.2 Risk factors ........................................................................................... 13 1.3.3 Inflammation ........................................................................................ 17 1.4 MicroRNAs ............................................................................................. 20 1.4.1 miRNA Biogenesis ............................................................................... 21 1.4.2 IsomiRs ................................................................................................. 25 1.4.3 RNA Editing ......................................................................................... 26 1.4.4 Non-templated nucleotide addition ...................................................... 27 1.4.5 miRNA turnover ................................................................................... 29 1.4.6 Post-translational modification of AGO .............................................. 31 1.4.7 Regulation of miRNAs by sequestration.............................................. 32 1.4.8 miRNA transport from the cytoplasm .................................................. 35 1.4.9 miR-140 ................................................................................................ 36 1.5 Immunological off-target effects following transfection of RNA sequences .............................................................................................................. 37 2 Aims ....................................................................................................................... 39 3 Summary of papers ................................................................................................ 40 4 Methodological considerations .............................................................................. 42 4.1 Patient material and sample processing ................................................... 42 4.2 Transfection via electroporation .............................................................. 42 4.3 Reverse transcription quantitative real-time polymerase chain reaction. 43 4.4 Western blot ............................................................................................. 44 4.5 Proteomics ............................................................................................... 45 4.6 RNA sequencing ...................................................................................... 49 5 Discussion .............................................................................................................. 50 5.1 Paper I ...................................................................................................... 50 5.2 Paper II .................................................................................................... 54 5.3 Paper III ................................................................................................... 57 6 Concluding remarks and future perspectives ........................................................ 61 References ................................................................................................................ 63 Acknowledgment The work presented in this thesis was performed at the Norwegian Center for Stem Cell Research, Department of Immunology, and the Department of Orthopedics, Institute of Clinical Medicine and the Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo and Oslo University Hospital – Rikshospitalet and Ullevål during the period of March 2015 to November 2020. This work was funded by South-Eastern Norway Regional Health Authority, UiO Innovation Funds and the Olav Thon Foundation. First of all I would like thank my supervisors Jan E. Brinchmann and Tommy A. Karlsen. I am grateful to Jan Brinchmann for recruiting me and giving me an extraordinary opportunity in a group that I have come to cherish after performing my Master's research there. I am also grateful for his abilities to secure funding when the funds and time ran out in order for me to complete this journey that helped relief the pressure tremendously. I am grateful to Tommy A. Karlsen for being the resourceful, knowledgeable, and encouraging person he is. His patience, pedagogical skills, ability to troubleshoot, and always having a back-up plan are inspiring. I am deeply indebted to his immense support which I wouldn't have been able to pull through without. I am privileged to have had the chance of knowing both and have them as my supervisors. I'm grateful for the time they have dedicated to supervise me and support me when I needed. A special thanks to Professor Jan Øivind Moskaug who sadly passed away in 2018, for his supervision with the mice pilot studies. Although the in vivo studies are not included in this thesis, his experience and feedback on application letters to Mattilsynet, and the techniques he taught me are invaluable. You are missed. I am also grateful to his student and my friend Annika Winsnes for being such a calm and pedagogical guide when handling the animals. I would like to acknowledge all members of my group for providing a harmonic, 1 supportive, and fun environment. All of you being around carried me through the toughest times of this journey. Thank you all former and present colleagues: Nadine Frerker, Krisztina Szöke, Mia-Elisabeth Rambøl, Kristin Flatmoen, Jim Eero Lamppu, Chloe Rixon, Barbora Hanousková, Torill Høiby, Rune B. Jakobsen, Biljana Stangeland, Åse-Karine Fjellheim, Karen Johanne Beckstrøm, Jardar Hinnaland Stendal, Kristian Lian and Magnus Østgård Olderøy. I am infinitely grateful to my family; my mother Magda, my father Nader, and my brother Mohammed for their unconditional love and infinite support I have always received, especially during this journey. I would not have made it without your love and encouragement. You are my rock! My aunts Afaf and Siham you've been my beacons, now your sweet memory and immense love will continue to shine. I thank all my friends near and far for being the wonderful people you are, for enriching my life, and for supporting me through this journey. A special gratitude to those who had been fellow comrades in the PhD-journey and always knew exactly what to say to unload some of the emotional burden of it; thank you Sarah Josefsson, Louise F. Risnes and Sarah F. Leuchtgens, Simen Antonsen and Mai Ziyada. Sarah J. your sense of responsibility, wisdom, and self-reflection are inspiring and liberating! Louise, I sincerely appreciate your valuable feedback on layout and proofreading of the thesis! Also a big thanks to Attila Zabo, Saja Salah, Guro Nærdal, Nermin Zecic, Sunniva Grønning, Victoria Nordhagen, and Ane Høiseth for being such good listeners and for being so supportive. Last, but not least, I would like to thank Rinzai-Zen senter, Oslo, for providing a place for reflection, introspection, and self-discovery. You have been indispensible during these past two years of the journey which were the toughest time. I'm forever indebted to you for being the oasis I take refuge in and for giving me the strength I needed to complete this journey. A special thanks to Christopher Cain, Jan Gaensslen, Leni Torvbråten and Christian Meaas Svendsen. Oslo, November 2020 Rua Nader Al-Modawi 2 Abbreviations AC articular cartilage ACAN aggrecan ACs articular chondrocytes ACI autologous chondrocyte implantation ACL articular crusiate ligament ADAR adenosine deaminase acting on RNA AGO argonaute ADAMTS disintegrin and metalloproteinase with thrombospondin motifs AQ absolute quantification ceRNA competing endogenous RNA CCL C-C motif chemokine ligand CIITA class II major histocompatibility complex transactivator circRNA circular RNA COL2A1 type II collagen CXCL C-X-C motif chemokine