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Characterization of ZNF281 and Ist Role in Colorectal Carcinogenesis

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Characterization of ZNF281 and Ist Role in Colorectal Carcinogenesis Aus dem Pathologischen Institut der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Thomas Kirchner In der Arbeitsgruppe Experimentelle und Molekulare Pathologie Leiter: Prof. Dr. rer. nat. Heiko Hermeking Characterization of ZNF281 and its Role in Colorectal Carcinogenesis Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Stefanie Hahn aus Weimar 2014 Gedruckt mit der Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Erstgutachter: Prof. Dr. rer. nat. Heiko Hermeking Zweitgutachter: Prof. Dr. rer. nat. Peter Nelson Dekan: Prof. Dr. med. Dr. h. c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung: 06.11.2014 I Meiner Familie. II EIDESSTATTLICHE VERSICHERUNG Stefanie Hahn Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema „Characterization of ZNF281 and its Role in Colorectal Carcinogenesis” selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. Ort, Datum:____________________ Unterschrift:_____________ III PUBLICATIONS Parts of this thesis have been published in: Original article: • Hahn S, Jackstadt R, Siemens H, Huenten S, Hermeking H (2013) SNAIL and miR-34a feed-forward regulation of ZNF281/ZBP99 promotes epithelial-mesenchymal transition. EMBO J 32: 3079-3095 Impact factor 2012 (ISI, Thomson Reuters): 9.8 Review article: • Hahn S and Hermeking H (2014) ZNF281/ZBP-99: a new player in epithelial-mesenchymal-transition, stemness and cancer. Journal of Molecular Medicine, revised version submitted Impact factor 2012 (ISI, Thomson Reuters): 4.8 IV ABBREVIATIONS Ago argonaute protein APC adenomatous polyposis coli APS ammonium peroxodisulfate ATM ataxia telangiectasia mutated ATR ATM and Rad3-related kinases bHLH-(LZ) basic helix-loop-helix (leucine zipper) bp base pair(s) CCSC colorectal cancer stem cell CD133 prominin 1 CDK cyclin-dependent kinase cDNA complementary DNA (q)ChIP (quantitative) chromatin immunoprecipitation c-MYC v-MYC avian myelocytomatosis viral oncogene homologue CpG cytidine-phosphate-guanidin CRC colorectal cancer CSC cancer stem cell Cy3 cyanine 3 DAPI 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride DMEM Dulbecco`s modified Eagles medium DMSO dimethyl-sulfoxide DNA deoxyribonucleic acid DNMT DNA-methyltransferase DOX doxycycline E.coli Escherichia coli E-box enhancer box EGF epidermal growth factor eGFP enhanced green fluorescent protein EMT epithelial-mesenchymal transition EMT-TF epithelial-mesenchymal transition transcription factor ESC embryonic stem cell FACS fluorescence-activated cell sorting FCS fetal calf serum FGF fibroblast growth factor FOXO3 forkhead box O3 GAPDH glyceraldehyde 3-phosphate dehydrogenase gDNA genomic DNA GSK3β glycogen synthase kinase-3β GZP1 GC-box-binding zinc-finger protein (ZNF281) HBSS Hank’s balanced salt solution HDAC histone deacetylase HIF-1α hypoxia-inducible factor-1α HDF human diploid fibroblast HGF hepatocyte growth factor hMSC human multiple stem cell HRP horseradish peroxidase V IF immunofluorescence IgG immunoglobulin INR initiator region IP immunoprecipitation kbp kilo base pairs LB lysogeny broth LEF1 lymphoid enhancer-binding factor 1 LGR5 leucine-rich repeat containing G protein-coupled receptor 5 LOX lysyl oxidase MAPK mitogen-activated protein kinase MAX MYC associated factor X MB MYC homology box MDCK cell Madin-Darby canine kidney cell MET mesenchymal-epithelial transition miR(NA) microRNA MIZ1 MYC-interacting zinc finger protein-1 mRNA messenger RNA mRFP monomer red fluorescent protein NICD Notch-intracellular domain ODC ornithine decaboxylase ORF open reading frame PAGE polyacrylamide gel electrophoresis P/C phase constrast PBS phosphate buffered saline (q)PCR (quantitative) polymerase chain reaction PDGF-β platelet-derived growth factor-β PDGFR platelet-derived growth factor receptor PI3K phosphoinositide-3-kinase PI propidium iodide polyHEMA poly(2-hydroxyethyl methacrylate) PP2A protein phosphatase 2A pri-miR(NA) primary microRNA RISC RNA induced silencing complex RNA ribonucleic acid RNAPol II RNA polymerase II ROS reactive oxygen species RT room temperature RTK receptor tyrosine kinase SD standard deviation SDS sodium dodecyl sulfate siRNA small interfering RNA TCF4 T-cell factor 4 temed tetramethylethylenediamine TF transcription factor TGF-β transforming growth factor-β TIC tumor initiating cell TRIS tris(hydroxymethyl)-aminomethan TSS transcription start site VI TWIST twist family bHLH transcription factor 1 UTR untranslated region VSV vesicular stomatitis virus (tag) WB Western blot WNT wingless-related integration site ZEB zinc finger E-box-binding homeobox protein ZNF281 zinc finger protein 281 VII TABLE OF CONTENTS Eidesstattliche Versicherung ................................................................................ III Publications ............................................................................................................ IV Abbreviations .......................................................................................................... V Table of contents ................................................................................................. VIII 1. Introduction ......................................................................................................... 1 1.1 The c-MYC oncogene ...................................................................................... 1 1.1.1 c-MYC and cancer ..................................................................................... 2 1.2 ZNF281/ZBP-99 .............................................................................................. 3 1.3 Epithelial-mesenchymal transition (EMT) ......................................................... 8 1.3.1 EMT and cancer ......................................................................................... 8 1.3.1.1 Molecular regulation of EMT ...................................................................... 8 1.3.1.2 EMT in cancer progression and the invasion-metastasis cascade . 12 1.4 microRNAs .................................................................................................... 15 1.4.1 The miR-34 family: members, regulation and the role in cancer ........ 17 2. Aims of the study .............................................................................................. 20 3. Materials ............................................................................................................ 21 3.1 Chemicals and reagents ................................................................................ 21 3.2 Enzymes ....................................................................................................... 22 3.3 Kits ................................................................................................................ 23 3.4 Antibodies ..................................................................................................... 23 3.4.1 Primary antibodies ................................................................................... 23 3.4.2 Secondary antibodies ................................................................................. 24 3.5 Vectors and oligonucleotides ......................................................................... 24 3.5.1 Vectors ...................................................................................................... 24 3.5.2 Oligonucleotides ...................................................................................... 26 3.5.2.1 Oligonucleotides used for qChIP ......................................................... 26 3.5.2.2 Oligonucleotides used for qPCR ......................................................... 27 3.5.2.3 Oligonucleotides used for cloning and mutagenesis ........................ 28 3.5.3 microRNA mimics and antagomiRs........................................................ 28 3.6 Buffers and solutions ..................................................................................... 29 3.7 Laboratory equipment .................................................................................... 31 4. Methods ............................................................................................................. 33 4.1 Bacterial cell culture ...................................................................................... 33 4.1.1 Propagation and seeding ........................................................................ 33 4.1.2 Transformation ......................................................................................... 33 4.1.3 Purification of plasmid DNA from E.coli ................................................ 33 VIII 4.2 Chromatin immunoprecipitation (ChIP) assay ................................................ 34 4.3 Cell culture of human cells ............................................................................. 35 4.3.1 Propagation of human cell lines ............................................................. 35 4.3.2 Transfection of oligonucleotides and vector constructs
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