Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | V, 148 S. Ill., graph. Darst. 30 cm |
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Datensatz im Suchindex
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| adam_text | CONTENTS I
CONTENTS
1 SUMMARY 1
2 INTRODUCTION 3
2.1 COLORECTAL CANCER .....3
2.1.1 MOLECULAR CAUSES AND PRIMARY TUMOR DEVELOPMENT 3
2.1.2 METASTASIS FORMATION 4
2.2 INTESTINAL EPITHELIUM 4
2.2.1 DIFFERENTIATED CELL TYPES IN THE INTESTINE 5
2.2.2 INTESTINAL STEM CELLS 6
2.2.3 INTESTINAL STEM CELL MARKERS 6
2.2.4 INTESTINAL STEM CELLS AS THE CELLS OF ORIGIN FOR CRC 7
2.2.5 SIGNALING PATHWAYS IMPORTANT FOR INTESTINAL HOMEOSTASIS 7
2.2.6 THE WNT/P-CATENIN SIGNALING PATHWAY 8
2.3 THE EPH FAMILY OF RECEPTOR TYROSINE KINASES 10
2.3.1 EPH RECEPTOR DOMAIN ORGANIZATION AND SIGNALING 11
2.3.2 CROSS-TALK WITH CELL SURFACE PROTEINS 13
2.3.3 EPH RECEPTORS IN PHYSIOLOGY 13
2.3.4 EPH RECEPTORS IN CANCER 14
2.3.5 EPHB RECEPTORS AND EPHRIN-B LIGANDS IN THE INTESTINE 14
2.3.6 EPHB RECEPTORS IN COLORECTAL CANCER 15
2.4 EPITHELIAL-MESENCHYMAL TRANSITION (EMT) 16
2.4.1 EMT INDUCERS 17
2.4.2 EMT AND STERNNESS . 18
2.4.3 REGULATION OF EMT INDUCERS 18
2.4.4 THE EMT INDUCER SNAIL1 19
2.4.5 COREPRESSOR COMPLEXES OF SNAIL1 20
2.5 EPIGENETIC GENE REGULATION 20
2.5.1 CHROMATIN 21
2.5.2 HISTONE MODIFICATIONS 21
2.5.3 DNA METHYLATION 22
2.5.4 ENHANCER CHROMATIN 23
HTTP://D-NB.INFO/1044588357
CONTENTS II
2.5.5 CANCER EPIGENETICS 23
3 AIM OF THE STUDY 25
4 MATERIALS 26
4.1 EQUIPMENT 26
4.2 CONSUMABLES 27
4.3 CHEMICALS AND REAGENTS 27
4.3.1 INHIBITORS 28
4.4 ENZYMES 28
4.5 BUFFERS AND SOLUTIONS 29
4.6 KITS ... 31
4.7 CELL CULTURE MEDIUM ! 31
4.8 HUMAN EUKARYOTIC CELL LINES 32
4.9 ANTIBODIES 32
4.9.1 PRIMARY ANTIBODIES - USED FOR CHIP AND CO-IMMUNOPRECIPITATION 32
4.9.2 PRIMARY ANTIBODIES - USED FOR WESTERN BLOT AND
IMMUNOHISTOCHEMISTRY 33
4.9.3 SECONDARY ANTIBODIES 33
4.10 OLIGONUCLEOTIDES 34
4.10.1 PRIMER FOR RT-PCR ANALYSES 34
4.10.2 PRIMER FOR CHIP AND FAIRE ANALYSES 35
4.10.3 PRIMER FOR CLONING 36
4.10.4 PRIMER FOR MUTAGENESIS 36
4.10.5 SIRNA OLIGONUCLEOTIDES 36
4.10.6 OLIGONUCLEOTIDES FOR EMSA 36
4.11 PLASMIDS 37
4.12 EDV 38
5 METHODS . 39
5.1 PLASMID CONSTRUCTION AND CLONING 39
5.1.1 MUTAGENESIS OF LUCIFERASE REPORTER CONSTRUCTS 40
5.2 TRANSFORMATION OF PLASMID DNA 40
CONTENTS III
5.3 ISOLATION OF PLASMID DNA 40
5.4 CELL CULTURE 41
5.5 GENERATION OF STABLE CELL LINES 41
5.6 INHIBITOR TREATMENT OF CELL LINES 44
5.7 ANALYSIS OF POPULATION DYNAMICS 44
5.8 ANALYSIS OF CELL MIGRATION 44
5.8.1 SCRATCH ASSAY 44
5.8.2 XCELLIGENCE* SYSTEM 45
5.9 SPHEROID FORMATION 45
5.10 RNA INTERFERENCE 45
5.11 LUCIFERASE ASSAY 46
5.12 AGAROSE GEL ELECTROPHORESIS 46
5.13 CHROMATIN IMMUNOPRECIPITATION (CHIP) 47
5.13.1 CHROMATIN PREPARATION.... 47
5.13.2 IMMUNOPRECIPITATION 47
5.13.3 DETERMINATION OF SHEARING EFFICIENCY 48
5.14 FORMALDEHYDE-ASSISTED ISOLATION OF REGULATORY ELEMENTS (FAIRE) 48
5.15 METHODS FOR WORKING WITH RNA 49
5.15.1 ISOLATION OF TOTAL RNA 49
5.15.2 DETERMINATION OF RNA CONCENTRATION 49
5.15.3 CDNA SYNTHESIS 49
5.16 POLYMERASE CHAIN REACTION (PCR) 50
5.16.1 SEMI-QUANTITATIVE REVERSE-TRANSCRIPTION (RT) - PCR 50
5.16.2 QUANTITATIVE REAL-TIME RT-PCR (QRT-PCR) 50
5.17 METHODS FOR WORKING WITH PROTEINS 52
5.17.1 PREPARATION OF WHOLE CELL LYSATES 52
5.17.2 QUANTIFICATION OF PROTEIN CONCENTRATION 52
5.17.3 CO-LMMUNOPRECIPITATION 52
5.17.4 SDS-POLYACRYLAMIDE-GEL ELECTROPHORESIS (SDS-PAGE) 52
5.17.5 WESTERN BLOT AND IMMUNODETECTION 53
CONTENTS IV
5.18 ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) 53
5.19 TISSUE SPECIMENS 54
5.19.1 IMMUNOHISTOCHEMISTRY 54
5.19.2 RNA ISOLATION, CDNA SYNTHESIS AND QPCR ANALYSIS OF FFPE HUMAN
TISSUE SPECIMENS 54
5.20 PAIRWISE CORRELATION ANALYSES OF RNA-SEQ EXPRESSION DATA 55
5.21 DATA PRESENTATION AND STATISTICAL ANALYSES 55
6 RESULTS 56
6.1 DIFFERENTIAL EXPRESSION OF THE WNT/P-CATENIN TARGET GENES EPHB2,
EPHB3
AND EPHB4 IN CRC 56
6.1.1 DIFFERENTIAL EXPRESSION OF EPHB2, EPHB3 AND EPHB4 IN CRC CELL
LINES 59
6.2 HDACS ARE INVOLVED IN DEREGULATION OF EPHB3 IN CRC CELL LINES 60
6.3 INVERSE EXPRESSION OF EPHB3 AND EMT INDUCERS IN CRC CELL LINES AND
PRIMARY
TUMORS 64
6.4 A PROMOTER-DISTAL ENHANCER OF EPHB3 HARBORS BINDING SITES FOR EMT
INDUCERS . 67
6.5 REGULATION OF EPHB3 BY EMT INDUCERS IN VITRO 69
6.5.1 REPRESSION OF EPHB3 BY SNAILL AND SNAIL2 IN VITRO 69
6.5.2 ZEB1 DOES
NOT AFFECT EPHB3 LUCIFERASE REPORTER CONSTRUCTS 71
6.6 REPRESSION OF ENDOGENOUS EPHB3 BY SNAILL 72
6.6.1 GENERATION OF CELL LINES WITH STABLE, DOX-INDUCIBLE EXPRESSION OF
SNAILL 72
6.6.2 EPHB3 IS A NOVEL AND DIRECT TARGET GENE OF SNAILL 73
6.7 MECHANISM OF EPHB3 REPRESSION BY SNAILL 76
6.7.1 SNAILL MEDIATES REPRESSION OF EPHB3 BY COUNTERACTING ASCL2 76
6.7.2 THE SNAG DOMAIN OF SNAILL IS IMPORTANT FOR REPRESSION OF EPHB3 79
6.7.3 IDENTIFICATION OF COREPRESSORS INTERACTING WITH THE SNAG DOMAIN OF
SNAILL 82
6.7.4 SNAILL EXPRESSION RESULTS IN CLOSURE OF EPHB3 ENHANCER CHROMATIN
88
6.8 SNAILL INDUCES EMT FEATURES IN LS174T CRC CELLS .90
6.8.1 TARGETING HDACS AND LSD1 REDUCES INVASIVE GROWTH IN 3D 92
6.9 INDUCTION OF SNAILL INTERFERES WITH THE INTESTINAL IDENTITY OF
LS174T CELLS 93
6.10 SUSTAINED EXPRESSION OF EPHB3 INTERFERES WITH SNAILL-INDUCED EMT 95
CONTENTS V
7 DISCUSSION.... 98
7.1 BIPHASIC, BUT NON-COORDINATED EXPRESSION PROFILES OF EPHB RECEPTORS
DURING
COLORECTAL TUMOR PROGRESSION 99
7.1.1 DEFINING A MODEL SYSTEM TO STUDY DIFFERENTIAL EXPRESSION OF EPHB
RECEPTORS 100
7.2 EPHB3 IS A NOVEL AND DIRECT TARGET GENE OF THE EMT INDUCER SNAILL
101
7.3 MECHANISM OF EPHB3 REPRESSION BY SNAILL 102
7.3.1 REPRESSION OF EPHB2-4 IS NOT CAUSED BY PROMOTER DNA
HYPERMETHYLATION IN CRC 103
7.3.2 INVOLVEMENT OF CLASS I HDACS IN THE REPRESSION OF EPHB3 103
7.3.3 INVOLVEMENT OF LSD1 IN THE REPRESSION OF EPHB3 105
7.3.4 COMPETITIVE DISPLACEMENT OF ASCL2 FROM THE EPHB3 ENHANCER 106
7.4 REPRESSION BY A MULTIPRONGED ATTACK ON THE EPHB3 ENHANCER 106
7.4.1 SNAILL INDUCES THE COLLAPSE OF OPEN CHROMATIN STRUCTURE AT THE
EPHB3 ENHANCER 108
7.5 RELEVANCE OF EMT IN CRC 110
7.5.1 SNAILL INDUCES EMT FEATURES IN LS174T CRC CELLS ILL
7.5.2 SNAILL INTERFERES WITH THE INTESTINAL IDENTITY OF LS174T CRC
CELLS ILL
7.5.3 SNAILL INDUCES EXPRESSION OF EPHRIN-B LIGANDS IN LS174T CRC CELLS
113
7.5.4 SNAILL TARGET GENES SHOW DIVERSE TRANSCRIPTIONAL RESPONSES 113
7.6 EPHB3 FUNCTIONS AS A ROADBLOCK AT THE ONSET OF EMT IN LS174T CRC
CELLS 114
8 BIBLIOGRAPHY 116
9 TABLE OF FIGURES 133
10 APPENDIX 135
10.1 ABBREVIATIONS 135
10.2 SUPPLEMENTARY INFORMATION 139
10.3 ACKNOWLEDGMENT 146
10.4 PUBLICATIONS 147
10.5 CURRICULUM VITAE 148
|
| any_adam_object | 1 |
| author | Rönsch, Kerstin 1984- |
| author_GND | (DE-588)1044588268 |
| author_facet | Rönsch, Kerstin 1984- |
| author_role | aut |
| author_sort | Rönsch, Kerstin 1984- |
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| bvnumber | BV041564303 |
| ctrlnum | (OCoLC)865732860 (DE-599)DNB1044588357 |
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| dewey-hundreds | 600 - Technology (Applied sciences) 500 - Natural sciences and mathematics |
| dewey-ones | 610 - Medicine and health 570 - Biology |
| dewey-raw | 610 570 |
| dewey-search | 610 570 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health 570 - Biology |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| spelling | Rönsch, Kerstin 1984- Verfasser (DE-588)1044588268 aut Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer vorgelegt von Kerstin Rönsch 2013 V, 148 S. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027009842&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rönsch, Kerstin 1984- Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer |
| subject_GND | (DE-588)4113937-9 |
| title | Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer |
| title_auth | Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer |
| title_exact_search | Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer |
| title_full | Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer vorgelegt von Kerstin Rönsch |
| title_fullStr | Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer vorgelegt von Kerstin Rönsch |
| title_full_unstemmed | Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer vorgelegt von Kerstin Rönsch |
| title_short | Snail1, an inducer of epithelial-mesenchymal transition, represses the EPHB3 tumor suppressor gene in colorectal cancer |
| title_sort | snail1 an inducer of epithelial mesenchymal transition represses the ephb3 tumor suppressor gene in colorectal cancer |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027009842&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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