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Mechanism of Microrna Mir-520G Pathogenesis in CNS-PNET

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Mechanism of Microrna Mir-520G Pathogenesis in CNS-PNET Mechanism of MicroRNA miR-520g Pathogenesis in CNS-PNET by J. H. David Shih A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by J. H. David Shih (2011) Mechanism of microRNA miR-520g pathogenesis in CNS-PNET J. H. David Shih Master of Science Graduate Department of Laboratory Medicine and Pathobiology University of Toronto 2011 Abstract We recently discovered a high-level amplicon spanning the chr19q13.41 microRNA cluster in CNS Primitive Neuroectodermal Tumour, which results in striking upregulation of miR-520g. Constitutive over-expression of miR-520g in untransformed human neural stem cells enhanced cell growth, restricted differentiation down the neuronal lineage, and promoted expression of neural stem/progenitor cell markers. We thus hypothesize that ectopic miR-520g expression promotes tumourigenesis in part by inhibiting cellular differentiation. Consistent with this proposition, miR-520g is silenced upon embryonic stem cell differentiation and its expression is absent from most adult tissues. Moreover, expression analysis of miR-520g overexpressing cells revealed significant dysregulation of developmental signalling pathways. Further efforts focused on elucidating mechanisms of miR-520g function led to the identification of a cell cycle inhibitor, p21, as an important candidate target. These findings collectively suggest that miR- 520g may modulate differentiation by regulating developmental signalling pathways and cell cycle exit of neural stem/progenitor cells. ii Acknowledgments I am thankful to my supervisor, Dr. Annie Huang, and my graduate committee members, Dr. Peter Dirks and Dr. Rod Bremner, whose encouragement, guidance, and support from the initial to the final stages of the project have provided a productive research environment and helped me develop an understanding of the subject. I am grateful for helpful discussions and feedbacks from Dr. Burton Yang and Dr. George M. Yousef on this thesis. I wish to express my gratitude to past and present members of the Huang Lab – Daniel Picard, Dr. Limei Zhou, Mohamed Ahmed, Dr. Meihua Li, Dr. Yuntao Lu, and Kyle F. Lee – for their advice and support during the course of my research. I am also grateful for helpful discussions with and advices from members of other labs. I would like to thank: Stephen Mack, Dr. Paul A. Northcott, and Adrian Dubuc from Dr. Michael Taylor’s lab for advice on quantitative PCR and molecular cloning; Sameer Agnihotri and Dr. Joydeep Mukherjee from Dr. Abhijit Guha’s lab for advice on molecular cloning and immunoblotting; Micky Tsui from Dr. Herman Yeger’s lab for advice on cell culture; Arthur Ling from Dr. Stephen Girardin’s lab for advice on transfection and luciferase reporter assay; Dr. Kevin Donato and Dr. Melanie Gareau from Dr. Philip M. Sherman’s lab for advice on luciferase reporter assay; Jacky Chung from Dr. Meredith Irwin’s lab for advice on transfection; and Dr. Ian D. Clarke from Dr. Peter Dirks’s lab for advice on immunocytochemistry. The progression of my project is also facilitated by the collaboration with and the generous sharing of equipment and reagents from other labs; I am grateful to: Dr. Peter Dirks, Dr. Paul Boutros, Dr. Herman Yeger, Dr. Meredith Irwin, Dr. Michael Taylor, Dr. Peter K. Kim, Dr. Jane McGlade, and Dr. Philip M. Sherman. I would also like to acknowledge members of the open source community, whose ongoing contribution to bioinformatic tools have been instrumental in the analytical aspect of my research project. I owe my gratitude to a great number of developers, including the R development team, the Bioconductor development team, and authors of various packages therein. This work was supported in part by the University of Toronto Fellowship, Ontario Graduate Scholarship, and the Hospital for Sick Children Research Training Competition. iii Table of Contents List of Tables ................................................................................................................................ vii List of Figures.............................................................................................................................. viii List of Appendices ......................................................................................................................... ix Abbreviations.................................................................................................................................. x Chapter 1 Introduction............................................................................................................. 1 1.1 Overview ........................................................................................................................... 2 1.2 Pediatric brain tumours...................................................................................................... 3 1.3 Mammalian central nervous system development ............................................................ 3 1.3.1 Terminological premise .......................................................................................... 3 1.3.2 Cellular players in neurogenesis ............................................................................. 4 1.3.3 Radial glial cells...................................................................................................... 4 1.3.4 Signalling pathways involved in neuroectodermal induction and neurogenesis .... 5 1.4 CNS-PNET and classification of pediatric embryonal brain tumours .............................. 6 1.5 MicroRNA: its function and biogenesis.......................................................................... 10 1.6 Chromosome 19 MicroRNA Cluster............................................................................... 12 1.6.1 Regulation of C19MC expression......................................................................... 12 1.6.2 C19MC dysregulation in cancer ........................................................................... 13 1.7 Project Objectives............................................................................................................ 13 Chapter 2 Materials and Methods.......................................................................................... 15 2.1 Cell cultures..................................................................................................................... 16 2.2 Plasmids........................................................................................................................... 16 2.3 Western Immunoblotting................................................................................................. 17 2.4 Quantitative RT-PCR...................................................................................................... 18 2.5 Induction of apoptosis by serum starvation..................................................................... 18 iv 2.6 Retinoic acid induced differentiation of NCCIT cells..................................................... 19 2.7 Transient transfections..................................................................................................... 19 2.8 Luciferase reporter assays............................................................................................... 19 2.8.1 Rationale for luciferase reporter constructs and cell lines.................................... 20 2.8.2 Positive controls for luciferase reporter assay ...................................................... 20 2.8.3 Optimized conditions for luciferase reporter assay .............................................. 21 2.9 Analyses of miRNA expression data............................................................................... 22 2.10 Expression array profiling............................................................................................... 22 2.11 Expression data analysis.................................................................................................. 22 2.11.1 Normalization ....................................................................................................... 22 2.11.2 Differential expression.......................................................................................... 22 2.11.3 Enrichment analysis of tumour expression profiles.............................................. 22 2.11.4 KEGG pathway enrichment analysis of PFSK and hNSC expression profiles .... 23 2.12 miRNA target prediction algorithms............................................................................... 23 Chapter 3 Results................................................................................................................... 24 3.1 Endogenous expression pattern of miR-520g.................................................................. 25 3.1.1 Rationale ............................................................................................................... 25 3.1.2 Change in miR-520g and C19MC microRNAs expression upon differentiation of human embryonic stem cells ............................................................................ 26 3.1.3 Endogenous expression pattern of miR-520g and C19MC microRNAs across normal tissues........................................................................................................ 28 3.1.4 miR-520g expression changes during retinoic-acid induced differentiation of NCCIT cells .........................................................................................................
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