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Using Kaposi's Sarcoma-Associated Herpesvirus to Elucidate the Role of Cellular Micrornas in Endothelial Biology

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Using Kaposi's Sarcoma-Associated Herpesvirus to Elucidate the Role of Cellular Micrornas in Endothelial Biology Using Kaposi’s sarcoma-associated herpesvirus to elucidate the role of cellular microRNAs in endothelial biology. Gemma Elizabeth Margaret Bridge BA (Hons) University College London Submitted for the degree of Doctor of Philosophy 2013 1 Declaration I, Gemma Elizabeth Margaret Bridge, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed: Date: 2 Acknowledgements First and foremost I would like to thank my PhD supervisor Chris Boshoff for allowing me to follow this project into fascinating new areas and for the many opportunities for career development that he has provided. I would also like to thank the rest of the CRUK viral oncology group for their assistance throughout the course of my PhD. In particular, I would like to thank Vicky Emuss for her patience, support and guidance during the start of my PhD. Special thanks must also go to Dimitris Lagos, Fiona Gratrix, Juanma Funes, Leonid Nikitenko, Emanuela Cuomo and Ohad Yogev for experimental advice, scientific chats, and more importantly, plain old camaraderie. I’d also like to thank Adrian Harris and Esther Bridges for invaluable advice on endothelial assays and most importantly, Rui Monteiro and Roger Patient, without whom, none of the zebrafish experiments would have been possible. Thank you to my amazing friends and family for listening to my complaints and for making me smile. Finally, I cannot begin to explain how much I appreciate the limitless love and support that Tom has given me over the last few years. Thank you for pushing me to continue. 3 Dedication I would like to dedicate this work to my parents, Philip and Anne. Thank you for everything. 4 Abstract Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ- herpesvirus that is the etiologic agent of Kaposi sarcoma (KS). KS is an angioproliferative neoplasm composed of cells of endothelial origin. For the work described in this thesis, KSHV infection of endothelial cells was used as a tractable model to study the role of microRNAs (miRNAs) in endothelial cell biology. Previous work in the laboratory had identified miRNAs which are either upregulated or downregulated upon KSHV infection of lymphatic endothelial cells (LEC). Target prediction analysis of these miRNAs and cross-comparison of predicted targets with expression levels of pro- and anti-angiogenic genes following KSHV infection, revealed the predicted targeting of Delta-like 4 (DLL4) by the miR-30 family. DLL4 is significantly upregulated in KSHV-infected lymphatic endothelial cells (KLEC) whereas the miR-30 family is significantly downregulated. DLL4 is a membrane-bound ligand belonging to the Notch signalling family that plays a fundamental role in vascular development and angiogenesis. Targeting of DLL4 by miR-30b and miR-30c was confirmed by examining mRNA and protein expression following transfection of endothelial cells with miR-30 mimics and inhibitors or infection with miR-30-expressing lentiviruses. The exact target site within the DLL4 3’UTR was identified using a luciferase reporter assay and site-directed mutagenesis. Overexpression of miR-30b in endothelial cells led to increased vessel number and length in an in vitro model of sprouting angiogenesis. Microinjection of miR- 30 into zebrafish embryos resulted in suppression of dll4 and subsequent excessive sprouting of intersegmental vessels and reduction in dorsal aorta diameter. Use of a target protector against the miR-30 site within the dll4 3’UTR upregulated dll4 and synergised with Vegfa signalling knockdown to inhibit angiogenesis. Furthermore, restoration of miR-30b or miR-30c expression during KSHV infection attenuated viral induction of DLL4. Overall, the work presented in this thesis demonstrates that the miR-30 family targets DLL4 to regulate angiogenesis. 5 Table of Contents List of Figures ................................................................................................... 11 List of Tables..................................................................................................... 13 Abbreviations .................................................................................................... 14 Publications....................................................................................................... 23 Chapter 1. Introduction ................................................................................ 24 1.1 Kaposi sarcoma-associated herpesvirus ................................................. 24 1.1.1 Kaposi sarcoma ................................................................................. 24 1.1.2 Lymphoproliferative disorders ........................................................... 27 1.1.3 Viral life cycle .................................................................................... 28 1.1.4 Angiogenic gene expression in KLEC ............................................... 29 1.2 Angiogenesis ........................................................................................... 31 1.2.1 Initiation of sprouting ......................................................................... 31 1.2.2 Sprout elongation and lumen formation ............................................. 32 1.2.3 Vessel stabilisation and maturation ................................................... 32 1.2.4 Vascular remodelling ......................................................................... 33 1.3 Zebrafish development ............................................................................ 34 1.3.1 General embryonic development ....................................................... 34 1.3.2 Development of the vasculature ........................................................ 36 1.4 Notch signalling ....................................................................................... 42 1.4.1 Notch receptors and ligands .............................................................. 42 1.4.2 Notch signal transduction .................................................................. 42 1.4.3 Regulation of the Notch pathway ....................................................... 45 1.4.4 Role in development .......................................................................... 46 1.4.5 Role in angiogenesis ......................................................................... 47 1.5 microRNAs .............................................................................................. 55 1.5.1 Discovery and classification .............................................................. 56 1.5.2 Biogenesis ......................................................................................... 57 6 1.5.3 Target recognition ............................................................................. 59 1.5.4 Mechanisms of silencing ................................................................... 62 1.5.5 Role in angiogenesis ......................................................................... 63 1.5.6 MicroRNA expression in KLEC .......................................................... 65 1.5.7 Role in zebrafish development .......................................................... 66 1.6 Thesis aims ............................................................................................. 69 Chapter 2. Materials and Methods ............................................................... 71 2.1 Cell Culture .............................................................................................. 71 2.1.1 Lymphatic endothelial cells ................................................................ 71 2.1.2 Human umbilical vein endothelial cells .............................................. 71 2.1.3 Immortalised human fibroblasts ......................................................... 72 2.1.4 BCBL-1 cells ...................................................................................... 72 2.1.5 293T cells .......................................................................................... 73 2.1.6 Freezing and thawing cells ................................................................ 73 2.2 Transfection, virus production and infection ............................................ 73 2.2.1 DNA transfection of 293T cells .......................................................... 73 2.2.2 Lentivirus production and infection of LEC and HUVEC .................... 74 2.2.3 KSHV production and infection of LEC .............................................. 75 2.2.4 Transfection of LEC, HUVEC, or 293T cells with miRNA mimics, miRNA inhibitors or siRNA ......................................................................... 77 2.3 Molecular biology techniques .................................................................. 78 2.3.1 PCR cloning strategy ......................................................................... 78 2.3.2 Restriction enzyme digestion ............................................................. 82 2.3.3 Agarose gel electrophoresis .............................................................. 82 2.3.4 Agarose gel extraction ....................................................................... 83 2.3.5 Ligation .............................................................................................. 84 2.3.6 Bacterial transformation ..................................................................... 84 2.3.7 Plasmid purification ........................................................................... 85 7 2.3.8 Site
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