The Cellular Function of USP22 and Its Role in Tissue Maintenance and Tumor Formation
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The Cellular Function of USP22 and its Role in Tissue Maintenance and Tumor Formation Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen, within the doctoral program “Molecular Biology of Cells” of the Georg-August University School of Science (GAUSS) submitted by Robyn Laura Kosinsky from Emmerich am Rhein, Germany Göttingen, 2017 Thesis supervisor Prof. Steven A. Johnsen, Ph.D. Thesis committee Prof. Steven A. Johnsen, Ph.D. Clinic for General, Visceral and Pediatric Surgery University Medical Center Göttingen Göttingen, Germany Prof. Dr. rer. nat. Holger Reichardt Institute for Cellular and Molecular Immunology University of Göttingen Medical School Göttingen, Germany Prof. Dr. med. Heidi Hahn Institute for Human Genetics University Medical Center Göttingen Göttingen, Germany Date of oral examination: 17.02.2017 i Affidavit I hereby declare that the Ph.D. thesis entitled “The Cellular Function of USP22 and its Role in Tissue Maintenance and Tumor Formation” has been written independently and with no other sources and aids than quoted. Göttingen, 03.01.2017 _______________________________ Robyn Laura Kosinsky ii TABLE OF CONTENTS Table of contents .................................................................................................................iii Abbreviations ......................................................................................................................vii List of figures ..................................................................................................................... xvi List of tables....................................................................................................................... xix Abstract ...............................................................................................................................xx 1. Introduction ...................................................................................................................... 1 1.1 Epidemiology of colorectal cancer (CRC) ..................................................................... 1 1.2 Risk factors associated with CRC ................................................................................. 2 1.2.1 Diet and lifestyle .................................................................................................... 2 1.2.2 Inflammatory bowel diseases (IBDs) ...................................................................... 3 1.2.3 Genetic susceptibility ............................................................................................. 4 1.2.4 Genetic mutations .................................................................................................. 7 1.2.5 Epigenetic deregulation ......................................................................................... 9 1.3 The composition of the intestinal system .....................................................................12 1.4 The intestinal microbiota ..............................................................................................16 1.5 The intestinal immune defense and ulcerative colitis ...................................................17 1.6 Colorectal tumor progression .......................................................................................20 1.7 Colorectal cancer detection .........................................................................................22 1.8 Classification of CRC subtypes ...................................................................................23 1.8.1 Histopathological classification ..............................................................................23 1.8.2 Molecular classification .........................................................................................24 1.9 Colorectal cancer treatment.........................................................................................26 1.10 Heterogeneity of colorectal cancer ............................................................................28 1.11 Murine models for ulcerative colitis and colorectal cancer .........................................30 1.12 Ubiquitination and cancer ..........................................................................................32 iii 1.13 USP22 as a crucial player of CRC .............................................................................34 1.14 Implications of USP22 in intestinal cell differentiation (preliminary data) ....................37 1.15 Objectives of this study ..............................................................................................40 2. Materials and Methods ...................................................................................................42 2.1 Materials......................................................................................................................42 2.1.1 Technical devices .................................................................................................42 2.1.2 Consumables ........................................................................................................45 2.1.3 Chemicals and reagents .......................................................................................47 2.1.4 Cell culture ............................................................................................................52 2.1.5 Kits .......................................................................................................................53 2.1.6 Oligonucleotides ...................................................................................................54 2.1.7 Antibodies .............................................................................................................56 2.1.8 Buffers ..................................................................................................................57 2.1.9 Software and tools ................................................................................................59 2.2 Methods ......................................................................................................................60 2.2.1 Animal studies ......................................................................................................60 2.2.1.2 Tamoxifen injection .........................................................................................60 2.2.1.4 Stool guaiac test .............................................................................................61 2.2.1.5 Determination of disease activity index (DAI) ..................................................61 2.2.1.6 Tissue isolation ...............................................................................................62 2.2.1.7 Serum isolation ...............................................................................................62 2.2.1.8 Isolation of intestinal epithelial cells ................................................................62 2.2.1.9 X-gal staining of embryos ...............................................................................63 2.2.1.10 Preparation of mouse embryonic fibroblasts (MEFs) ....................................63 2.2.1.11 Preparation of paraffin-embedded tissue ......................................................64 2.2.1.12 H&E and Nissl staining .................................................................................64 2.2.1.13 Immunohistochemistry (IHC) ........................................................................65 2.2.1.14 Histo-score (H-score) ...................................................................................65 2.2.1.15 Mechanical bone testing ...............................................................................66 2.2.2 Cell culture ............................................................................................................66 2.2.2.1 Cell culture and inhibitor treatment .................................................................66 2.2.2.2 siRNA transfection ..........................................................................................66 2.2.2.3 Proliferation assessment ................................................................................67 2.2.2.4 Migration assay ..............................................................................................67 2.2.2.5 Colony formation assay ..................................................................................67 2.2.2.6 Soft agar colony formation assay ....................................................................68 2.2.2.7 CRISPR/Cas9-mediated knockout of USP22 ..................................................68 2.2.3 Molecular biology techniques ................................................................................69 2.2.3.1 DNA extraction from tail biopsies or cells ........................................................69 2.2.3.2 Genotyping of experimental mice ...................................................................69 iv 2.2.3.3 RNA isolation ..................................................................................................70 2.2.3.4 RNA gel electrophoresis .................................................................................70 2.2.3.5 cDNA synthesis ..............................................................................................70 2.2.3.6 Quantitative Real-Time PCR (qPCR) ..............................................................71 2.2.3.7 Luciferase reporter assay ...............................................................................71