Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Berlin
2016
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| Beschreibung: | 149 Blätter Illustrationen, Diagramme |
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| 245 | 1 | 0 | |a Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer |c von M.Sc. Katharina Ilm |
| 264 | 1 | |a Berlin |c 2016 | |
| 300 | |a 149 Blätter |b Illustrationen, Diagramme | ||
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| 502 | |b Dissertation |c Humboldt-Universität zu Berlin |d 2016 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 776 | 0 | 8 | |i Reproduziert als |a Ilm, Katharina |t Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer |d Berlin : MIK-Center GmbH, 2016 |h 2 Mikrofiches |w (DE-604)BV044489518 |
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Datensatz im Suchindex
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| adam_text | INDEX O F CONTENTS
INDEX OF CONTENTS
ABSTRACT 7
ZUSAMMENFASSUNG 8
1. INTRODUCTION 10
1.1 COLORECTAL CANCER: EPIDEMIOLOGY 10
1.1.1 INCIDENCE AND MORTALITY 10
1.1.2 RISK FACTORS AND HEREDITARY CAUSES FOR CRC 10
1.2 TUMORIGENESIS AND PROGRESSION OF CRC 11
1.2.1 ANATOMY AND HISTOLOGY OF THE LARGE INTESTINE 11
1.2.2 MALIGNANT TRANSFORMATION OF COLONIC EPITHELIAL CELLS AND STEP-WISE
PROGRESSION 12
1.3 MOLECULAR PATHWAYS INVOLVED IN CRC 13
1.3.1 CIN PATHWAY 13
1.3.2 MSI PATHWAY 18
1.3.3 CIMP PATHWAY 19
1.4 MICRORNAS - POST-TRANSCRIPTIONAL REGULATORS OF GENE EXPRESSION 19
1.4.1 BIOGENESIS AND FUNCTION OF MICRORNAS 20
1.4.2 MIRNA TARGET RECOGNITION 22
1.4.3 THE ROLE OF MIRNA IN CANCER ESPECIALLY IN CRC 24
1.5 METASTATIC SPREAD OF CRC 25
1.5.1 METASTATIC CASCADE 25
1.5.2 MOLECULAR PATHWAYS INVOLVED IN CRC METASTASIS 26
1.6 CLINICAL ASPECTS OF CRC 28
1.6.1 CRC STAGING AND PROGNOSIS 28
1.6.2 THERAPY FOR CRC 29
1.7 PROGNOSTIC AND PREDICTIVE MOLECULAR MARKERS OF CRC 30
1.7.1 KRAS 31
1.7.2 BRAF 31
1.7.3 MSI 31
1.8 MACC1 - A PROMISING PROGNOSTIC MARKER IN SOLID TUMORS 32
1.8.1 GENE STRUCTURE AND PROTEIN DOMAINS
1.8.2 TRANSCRIPTIONAL REGULATION OF MACC1 33
1.8.3 POST-TRANSCRIPTIONAL REGULATION OF MACC1 BY MIRNAS 34
1.8.4 ROLE OF MACC1 IN SIGNALING PATHWAYS 34
1.8.5 ROLE OF MACC1 IN TUM OR PROGRESSION AND METASTASIS 36
1.8.6 MACC1 AS PROMISING PROGNOSTIC AND PREDICTIVE MARKER FOR SOLID
CANCER 36
1.9 AIMS OF THE STUDY 38
2. MATERIAL AND METHODS 39
2.1 CLONING 39
2.1.1 CONSTRUCTION OF WILD TYPE MACCL-3 -UTR LUCIFERASE PLASMID 39
2.1.2 SITE-DIRECTED MUTATION OF MIR-218 SEED SEQUENCE IN THE
MACCL-3 -UTR VECTOR 40
2.2 BIOMARKER ANALYSES 40
2.2.1 PATIENTS CHARACTERISTICS 40
2.2.2 DNA AND RNA ISOLATION FROM PATIENT TISSUE 41
2.2.3 KRAS AND BRAF MUTATION ANALYSES 41
2.2.4 MICROSATELLITE INSTABILITY ANALYSIS 42
2.2.5 QUANTITATIVE REAL-TIME RT-PCR (QRT-PCR) OF CRC PATIENT TISSUE 42
2.3 CELL CULTIVATION, TRANSFECTIONS AND INHIBITOR TREATMENTS 43
2.3.1 CRC AND GASTRIC CANCER CELL LINES 43
2.3.2 TRANSFECTION OF EXPRESSION PLASMIDS 45
2.3.3 TRANSFECTION OF SIRNAS AND MIRNAS 46
2.3.4 TRANSFECTION OF MACC1 PROMOTER LUCIFERASE CONSTRUCT 47
2.3.5 TRANSFECTION OF DIFFERENT MACCL-3 -UTR DUAL LUCIFERASE CONSTRUCTS
47
2.3.6 INHIBITOR TREATM ENT 47
2.4 GENE EXPRESSION ANALYSIS 48
2.4.1 RNA ISOLATION, QUANTIFICATION AND QUALITY CONTROL 48
2.4.2 GENOME-WIDE EXPRESSION ANALYSIS (MICROARRAYS) 48
2.4.3 REVERSE TRANSCRIPTION 49
2.4.4 QRT-PCR 49
2.4.5 PROTEIN ISOLATION AND QUANTIFICATION 51
2.4.6 WESTERN BLOT ANALYSIS 51
2.5 FUNCTIONAL
IN VITRO
ASSAYS 52
2.5.1 DUAL LUCIFERASE REPORTER GENE ASSAY 52
2.5.2 BOYDEN CHAMBER MIGRATION AND INVASION ASSAY 52
2.5.3 COLONY FORMATION ASSAY 53
2.5.4 PROLIFERATION ASSAY 53
2.6 ALTERNATIVE POLYADENYLATION (APA) DETERMINATION (3 RACE EXPERIMENT)
53
2.7 STATISTICS
54
3. RESULTS 56
3.1 PROGNOSTIC MARKER ANALYSES 56
3.1.1 ASSOCIATIONS OF MACC1 EXPRESSION WITH CLINICOPATHOLOGICAL
PARAMETERS 56
3.1.2 ASSOCIATIONS OF MACC1 EXPRESSION WITH MSI STATUS, KRAS 612, KRAS
613 OR BRAF V600E
MUTATIONS 58
3.1.3 MFS PROGNOSIS ACCORDING TO MACC1 EXPRESSION, KRAS 612 OR 613
MUTATIONS, BRAF MUTATION
AND MSI STATUS 60
3.1.4 IMPACT OF CLINICOPATHOLOGICAL PARAMETERS ON MACC1 EXPRESSION OR
KRAS 613D MUTATION BASED
MFS PROGNOSES 63
3.1.5 IMPACT OF THE MOLECULAR MARKERS KRAS MUTATIONS, BRAF V600E
MUTATION AND MSI STATUS ON
MACC1 EXPRESSION BASED MFS PROGNOSIS 64
3.1.6 COMBINATORIAL SURVIVAL ANALYSIS OF THE INDEPENDENT PROGNOSTIC
MARKERS MACC1 EXPRESSION AND
KRAS 613D MUTATION 64
3.1.7 CLUSTER CLASSIFICATION 65
3.2 TRANSCRIPTIONAL REGULATION OF MACC1 BY KRAS 67
3.2.1 ECTOPIC OVEREXPRESSION OF DIFFERENT KRAS MUTATIONS IN KRAS WILD
TYPE CELL LINES HAD NO IMPACT
ON MACC1 EXPRESSION 67
3.2.2 KRAS KNOCK-DOWN IN 612V MUTATED CELL LINES HAS OPPOSITE EFFECTS ON
MACC1 EXPRESSION 68
3.2.3 KRAS KNOCK-DOWN IN 613D MUTATED CELL LINES INDUCES MACC1
EXPRESSION IN HCT116 BUT NOT IN
DLD-1 CELLS 69
3.2.4 KRAS KNOCK-DOWN HAD NO IMPACT ON MACC1 EXPRESSION IN LS174T AND
HT29 CELLS 70
3.2.5 DISRUPTION OF THE 613D MUTATED KRAS INDUCED MACC1 EXPRESSION IN
HCT116 CELLS 72
3.2.6 KNOCK-OUT OF THE KRAS 613D MUTATED ALLELE LED TO UPREGULATED IFN
SIGNALING WHICH MIGHT INDUCE
MACC1 EXPRESSION 74
3.2.7 INHIBITOR STUDIES FURTHER INDICATE INVOLVEMENT OF JAK/STAT
SIGNALING IN REGULATION OF MACC1
EXPRESSION 81
3.2.8 STAT 1 AND STAT2 KNOCK-DOWN DOWNREGULATES MACC1 EXPRESSION 85
3.3 POST-TRANSCRIPTIONAL REGULATION OF MACC1 BY MICRORNAS 86
3.3.1 MACC1 3 UTR HARBORS BINDING SITES FOR MIRNAS 86
3.3.2 MIR-218 EXPRESSION INVERSELY CORRELATED WITH MACC1 EXPRESSION IN
CRC
3.3.3 THE MACC1 3 UTR IS A TARGET FOR MIR-218 88
3.3.4 MIR-218 REGULATES MACC1 GENE EXPRESSION 90
3.3.5 MIR-218 INHIBITS MACCL-INDUCED MIGRATION, INVASION AND COLONY
FORMATION 91
3.3.6 MIR-218 INHIBITED MACC1 EXPRESSION AND ITS MEDIATED TUM OR
PROGRESSIVE EFFECTS IN GASTRIC
CANCER 94
3.3.7 MACC1 POSSESSES ALTERNATIVE POLYADENYLATION (APA) SITES 96
4. DISCUSSION 97
4.1 PROGNOSTIC MARKER ANALYSES 98
4.1.1. HIGH MACC1 EXPRESSION CORRELATES WITH METACHRONOUS METASTASES,
KRAS G13D MUTATION, WILD
TYPE BRAF AND MSS/MSI-L 98
4.1.2 HIGH MACC1 EXPRESSION AND KRAS G13D MUTATION ARE INDEPENDENT
PROGNOSTIC MARKER FOR
REDUCED MFS 100
4.1.3 COMBINATION OF HIGH MACC1 EXPRESSION AND KRAS G13D MUTATION ALLOWS
THE IDENTIFICATION OF
HIGH RISK PATIENTS FOR REDUCED MFS 102
4.1.4 REFINEMENT OF THE CRC CLASSIFICATION BY INCLUSION OF MACC1
EXPRESSION AND KRAS G13D
MUTATION 103
4.1.5 SUMMARY: THE ROLE OF THE PROGNOSTIC MARKER MACC1 IN MOLECULAR
SUBTYPES OF CRC 105
4.2 TRANSCRIPTIONAL REGULATION OF MACC1 106
4.2.1 EFFECTS ON MACC1 EXPRESSION INDUCED BY OVEREXPRESSION OR
KNOCK-DOWN OF KRAS WERE NOT
SPECIFIC FOR A CERTAIN KRAS MUTATION 107
4.2.2 STAT1 AND STAT2 ARE POSSIBLE TRANSCRIPTIONAL REGULATORS OF MACC1
109
4.2.3 SUMMARY: TRANSCRIPTIONAL REGULATION OF MACC1 BY DIFFERENT KRAS
MUTATIONS AND ITS NEW
POTENTIAL REGULATORS, STAT1 AND STAT2 112
4.3 POST-TRANSCRIPTIONAL REGULATION OF MACC1 BY MIRNAS 114
4.3.1 MACC1 3 UTR IS A POSSIBLE TARGET FOR TUM OR SUPPRESSIVE MIRS 115
4.3.2 MIR-218 EXPRESSION INVERSELY CORRELATES WITH MACC1 EXPRESSION IN
CRC CELLS 115
4.3.3 MIR-218 INHIBITS MACC1 EXPRESSION AND ITS MEDIATED TUM OR
PROGRESSIVE EFFECTS IN CRC AND
GASTRIC CANCER 116
4.3.4 MACC1 POSSESSES ALTERNATIVE POLYADENYLATION (APA) SITES 117
4.3.5 SUMMARY: POST-TRANSCRIPTIONAL REGULATION OF MACC1 BY MIRNAS 118
5. OUTLOOK 119
ABBREVIATIONS 121
REFERENCES 127
ERKLAERUNG
146
PUBLICATION LIST 147
ACKNOWLEDGEMENT 149
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| spelling | Ilm, Katharina Verfasser (DE-588)1137383461 aut Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer von M.Sc. Katharina Ilm Berlin 2016 149 Blätter Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Humboldt-Universität zu Berlin 2016 (DE-588)4113937-9 Hochschulschrift gnd-content Reproduziert als Ilm, Katharina Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer Berlin : MIK-Center GmbH, 2016 2 Mikrofiches (DE-604)BV044489518 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029827347&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ilm, Katharina Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer |
| subject_GND | (DE-588)4113937-9 |
| title | Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer |
| title_auth | Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer |
| title_exact_search | Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer |
| title_full | Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer von M.Sc. Katharina Ilm |
| title_fullStr | Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer von M.Sc. Katharina Ilm |
| title_full_unstemmed | Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer von M.Sc. Katharina Ilm |
| title_short | Transcriptional and post-transcriptional regulation of the metastasis associated gene MACC1 and its importance as prognostic marker in colorectal cancer |
| title_sort | transcriptional and post transcriptional regulation of the metastasis associated gene macc1 and its importance as prognostic marker in colorectal cancer |
| topic_facet | Hochschulschrift |
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