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Identification and Characterization of MYCN-Driven Neuroblastoma

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Identification and Characterization of MYCN-Driven Neuroblastoma Identification and characterization of microRNAs that play novel functional roles in MYCN-driven neuroblastoma Chi Yan Ooi A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy (Research) Children’s Cancer Institute School of Women’s and Children’s Health Faculty of Medicine UNSW Sydney November 2017 http://doi.org/10.4225/53/5a1b3688156e3 Thesis/Dissertation Sheet Thesis/Dissertation Sheet Chi Yan Ooi i Originality Statement Chi Yan Ooi ii Copyright and Authenticity Statements Chi Yan Ooi iii Table of Contents Table of Contents Thesis/Dissertation Sheet ...................................................................................................i Originality Statement ....................................................................................................... ii Copyright and Authenticity Statements .......................................................................... iii Table of Contents .............................................................................................................iv List of Figures ..................................................................................................................xi List of Tables ...................................................................................................................xv Abstract ..........................................................................................................................xvi Abbreviations .............................................................................................................. xviii Publication in preparation from this thesis .................................................................. xxiii Conference presentations arising from this thesis ........................................................xxiv Acknowledgements ......................................................................................................xxvi Introduction ....................................................................................................1 Cancer................................................................................................................2 1.1.1 Tumorigenesis – Initiation, Promotion and Progression of Cancer ............. 2 1.1.2 Oncogenes, Tumour Suppressors and the Hallmarks of Cancer ................. 3 Neuroblastoma ..................................................................................................6 1.2.1 Clinical Stages, Features and Management ................................................. 9 1.2.2 MYCN: A major proto-oncogene of neuroblastoma ................................. 14 MYCN and neuroblastoma initiation and progression ......................17 MYCN and the dysregulation of microRNA in neuroblastoma ........17 Chi Yan Ooi iv Table of Contents MicroRNA .......................................................................................................19 1.3.1 The biogenesis of microRNAs .................................................................. 19 1.3.2 miRNA binding sites on mRNAs .............................................................. 24 1.3.3 MicroRNA stability ................................................................................... 29 1.3.4 MicroRNA’s non-canonical function in positive regulation of translation ................................................................................................................ 30 1.3.5 MicroRNA’s non-canonical functions in the nucleus ............................... 31 MicroRNA and cancer: Acting as tumour suppressor or oncogene or both ...33 1.4.1 MicroRNA in neuroblastoma biology ....................................................... 34 The miR-17-92 cluster, its paralogs and target genes ........................35 1.4.1.1.1 MYCN-E2F1 negative feedback loop ..............................................37 1.4.1.1.2 Regulation of TGFβ pathway by the miR-17-92 cluster ..................40 1.4.1.1.3 Regulation of Cell cycle by the miR-17-92 cluster ..........................42 1.4.1.1.4 Regulation of estrogen receptor-α (ESR1) and neuroblast differentiation by the miR-17-92 cluster .........................................................44 The LIN28B-let-7-MYCN axis and neuroblastoma initiation ...........45 The roles of miR-34a in neuroblastoma.............................................50 The role of miR-9 in MYCN driven neuroblastoma ..........................54 miR-204 as a potential tumour suppressor of neuroblastoma ............57 miR-375’s interactions with MYCN ..................................................60 miRNAs that directly repress MYCN expression ..............................61 1.4.2 MicroRNAs as therapeutic targets in neuroblastoma ................................ 61 Chi Yan Ooi v Table of Contents 1.4.3 MicroRNAs as biomarkers for neuroblastoma .......................................... 64 Summary and prospective ...............................................................................66 Materials and Methods .................................................................................69 Bioinformatical analyses .................................................................................70 2.1.1 Enrichment Analysis with GeneCodis3 ..................................................... 70 2.1.2 Compute overlaps with gene sets from MSigDB ...................................... 70 2.1.3 miRNA targeting predictions..................................................................... 71 2.1.4 R2 Kaplan Meier by gene expression analysis .......................................... 72 2.1.5 Peer-reviewed high-throughput miRNA expression profiling studies of human neuroblastoma samples................................................................................ 73 Cell biology techniques ...................................................................................74 2.2.1 Cell lines and culture conditions ............................................................... 74 2.2.2 Cell viability counts ................................................................................... 74 2.2.3 Doxycycline-induced induction of MYCN and miR-204 over- expression ................................................................................................................ 75 2.2.4 Panobinostat and 5-Aza-2’-deoxycytidine treatments ............................... 75 Transfection of neuroblastoma cells ...............................................................76 2.3.1 Transient transfection of siRNA or miRNA mimic / inhibitor .................. 76 2.3.2 Stable transfection of lentiviral shMIMIC miRNA ................................... 79 Molecular biological techniques .....................................................................82 2.4.1 miRNA extraction...................................................................................... 82 2.4.2 TaqMan microRNA reverse transcription and assays ............................... 83 Chi Yan Ooi vi Table of Contents 2.4.3 mRNA extraction ....................................................................................... 86 2.4.4 cDNA synthesis and qPCR ........................................................................ 86 2.4.5 Whole cell protein extraction .................................................................... 89 2.4.6 Protein quantification ................................................................................ 89 2.4.7 Western blotting ........................................................................................ 90 2.4.8 Chromatin Immunoprecipitation Assay..................................................... 91 2.4.9 Biotin-labelled miRNA mimic pulldown of mRNAs ................................ 95 2.4.10 Microarray ................................................................................................. 97 Cell phenotype and functional assays .............................................................98 2.5.1 Cell viability and cell proliferation assays ................................................ 98 2.5.2 Colony forming assay ................................................................................ 99 2.5.3 Fluorescence microscopy ........................................................................ 100 Mouse models of neuroblastoma...................................................................101 2.6.1 Th-MYCN mouse neuroblastoma tumorigenesis model .......................... 101 Ganglia dissection ............................................................................101 2.6.2 Stable cells subcutaneous xenografts ...................................................... 102 Engraftment and monitoring ............................................................102 Doxycycline administration and in vivo fluorescence imaging .......103 Tumour tissue preparation ...............................................................104 Statistical analysis .........................................................................................105 Identification of candidate miRNAs & mRNAs for roles in neuroblastoma tumorigenesis ................................................................................................................106 Chi Yan Ooi vii Table of Contents Introduction ................................................................................................... 107 Results ........................................................................................................... 110 +/+ 3.2.1 Th-MYCN ganglia tissue profiling
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