University of Dundee DOCTOR OF PHILOSOPHY Epigenetic dysregulation of novel candidate genes in melanoma Wang, Hexiao Award date: 2013 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 04. Oct. 2021 DOCTOR OF PHILOSOPHY Epigenetic dysregulation of novel candidate genes in melanoma Hexiao Wang 2013 University of Dundee Conditions for Use and Duplication Copyright of this work belongs to the author unless otherwise identified in the body of the thesis. It is permitted to use and duplicate this work only for personal and non-commercial research, study or criticism/review. You must obtain prior written consent from the author for any other use. Any quotation from this thesis must be acknowledged using the normal academic conventions. It is not permitted to supply the whole or part of this thesis to any other person or to post the same on any website or other online location without the prior written consent of the author. Contact the Discovery team ([email protected]) with any queries about the use or acknowledgement of this work. Epigenetic dysregulation of novel candidate genes in melanoma Hexiao Wang BSc, MSc Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Supervisors: Professor Charlotte Proby, Dr Tim Crook, Dr Gareth Inman. August 2013 Table of Contents Table of Contents ................................................................................................................. I List of Figures .................................................................................................................... IV List of Tables ..................................................................................................................... VII Abbreviations ................................................................................................................... VIII Declaration ......................................................................................................................... XI Acknowledgements .......................................................................................................... XII Abstract ........................................................................................................................... XIV Aims ................................................................................................................................. XVI Chapter 1 Introduction ....................................................................................................... 1 1.1 Cutaneous Melanoma (CM) ....................................................................................... 2 1.1.1 Epidemiology of melanoma ................................................................................ 3 1.1.2 Histological progression of melanoma.............................................................. 4 1.1.3 Subtypes of CM ................................................................................................... 7 1.1.4 Staging system of melanoma ............................................................................. 7 1.1.5 Risk factors for melanoma ............................................................................... 13 1.2 Genetic and epigenetic aberrations of melanoma................................................. 19 1.2.1 Genetic dysregulation of melanoma ................................................................ 19 1.2.2 Targeted therapies for melanoma .................................................................... 26 1.2.3 Epigenetic dysregulation of melanoma ........................................................... 28 Chapter 2 Materials and Methods ................................................................................ 39 2.1 Cell lines and culture conditions ............................................................................ 40 2.1.1 Human melanocyte and melanoma cell culture .............................................. 40 2.1.2 Human melanoma cell line culture for microarray .......................................... 42 2.1.3 Disassociation, freezing and recovery of cells ............................................... 43 2.2 Tissue and serum samples ..................................................................................... 44 2.3 Molecular biology methods .................................................................................... 45 2.3.1 DNA and RNA Purification ................................................................................ 45 2.3.2 Reverse transcription of mRNA to cDNA ........................................................ 49 2.3.3 Quantitative real-time polymerase chain reaction (qPCR) ............................. 50 I | P a g e 2.3.4 DNA Methylation analysis ................................................................................ 53 2.4 Protein procedures .................................................................................................. 61 2.4.1 Western blotting ................................................................................................ 61 2.4.2 Immunofluorescence ........................................................................................ 66 2.5 Cell biology assays ................................................................................................. 67 2.5.1 Transient transfection of short interfering RNA (siRNA)................................ 67 2.5.2 Methylation reversal assay ............................................................................... 70 2.5.3 Hypoxia treatment ............................................................................................. 70 2.5.4 Cell counting ..................................................................................................... 71 2.6 Microarray analysis ................................................................................................. 71 2.6.1 Methylation reversal assay with expression microarray ................................ 71 2.6.2 Expression microarray ..................................................................................... 72 2.6.3 Methylation microarray ..................................................................................... 72 2.7 Statistics .................................................................................................................. 73 Chapter 3 Identification of candidate biomarkers by systematic approaches ............. 74 3.1 Introduction ............................................................................................................. 75 3.1.1 Tissue factor pathway inhibitor 2 (TFPI2) ........................................................ 77 3.1.2 Early growth response gene 2 (EGR2) ............................................................ 79 3.1.3 Dickkopf homolog 1 (DKK1) ............................................................................. 80 3.1.4 SMAD family member 3 (SMAD3) ..................................................................... 81 3.1.5 TSC22 domain family, member 1 (TSC22D1) .................................................. 82 3.2 Results ..................................................................................................................... 82 3.2.1 Identification of candidate genes by methylation reversal assay combined with gene expression profiling ................................................................................. 82 3.2.2 Identification of candidate genes by systematic methylation profiling and gene expression profiling in paired melanoma cell lines...................................... 111 3.3 Discussion ............................................................................................................. 117 Chapter 4 Methylation investigation of candidate genes NT5E, DUSP2, and Dab2. .. 123 4.1 Introduction ........................................................................................................... 124 4.1.1 NT5E (ecto-5’-nucleotidase, CD73) ................................................................ 124 4.1.2 DUSP2 (Dual-specificity phosphatase 2) ....................................................... 126 4.1.3 Dab2 (Disabled-2) ............................................................................................ 128 4.2 Results ................................................................................................................... 129 4.2.1 NT5E methylation in melanoma ..................................................................... 129 4.2.2 DUSP2 methylation in melanoma................................................................... 138 4.2.3 Dab2 methylation in melanoma ...................................................................... 146 II | P a g e 4.3 Discussion