Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 84 S. Ill., graph. Darst, 21 cm |
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| 245 | 1 | 0 | |a Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta |c vorgelegt von Jia Huang |
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| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
ABBREVIATIONS
1. INTRODUCTION 1
1.1 MLRNAS ARE DYSREGULATED IN
LIVER FIBROSIS AND
CARCINOGENESIS 1
1.1.1 BIOGENESIS OF MICRORNAS 2
1.1.2 DYSREGULATION OF MIRNAS DURING LIVER FIBROSIS AND MYOFIBROBLASTS
OF HSCS 3
1.1.3 THE DYSREGULATION OF MIR-29 IN ACTIVATED HEPATIC STELLATE CELLS
RESULTS IN ECM ACCUMULATION.. S
1.1.4 MIRNAS IS DYSREGULATED AND INVOLVED IN MOLECULAR ALTERATION OF HCC
6
1.1.5 MIR-29 FAMILY IS DYSREGULATED IN HCC 7
1.2 TGF-P REGULATES
GENE TRANSCRIPTION VIA DIFFERENT PATHWAYS 8
1.2.1 CANONICAL SMAD SIGNALING AFTER TGF-P STIMULATION 8
1.2.2 TAKI INDUCED P38/JNK MAPK SIGNALING 10
1.2.3 ERKL/2 MAPK PATHWAY INDUCED BY TGF-P 11
1.2.4 P13K/AKT PATHWAY INDUCED BY TGF-P 12
1.3 AIM OFT1IE
STUDY 14
2. MATERIALS AND METHODS 16
2.1 MATERIALS 16
2.1.1 SOFTWARE 16
2.1.2 ENZYMES AND ANTIBODIES 16
2.1.2.1 ENZYMES 16
2.1.2.2 ANTIBODIES 16
2.1.3 DEVICES 17
2.1.4 CELL CULTURE 17
2.1.5 REAGENTS 17
2.1.6 KITS AND ASSAYS 17
2.1.7 OLIGONUCLEOTIDES 18
2.1.8 PLASMIDS 20
HTTP://D-NB.INFO/1065415214
TABLE OF CONTENTS
2.1.9 BUFFERS AND SOLUTIONS 20
2.1.10 CELL LINES 23
2.1.11
OTHER MATERIALS
23
2.2 METHODS 24
2.2.1 CELL CULTURE 24
2.2.1.1 STIMULATION 24
2.2.1.2 PASSAGE OF CELLS 24
2.2.1.3 CRYOPRESCRVATION OF CELLS 24
2.2.1.4 TRANSFCCTION OF PLASMID DNA 25
2.2.2 DNA PREPARATION 25
2.2.2.1 DNA PREPARATION OF E. COLI 25
2.2.2.2 DNA EXTRACTION BY USING PROTEINASE K AND PHENOL 26
2.2.2.3 ELECTROPHORETIC SEPARATION OF DNA 26
2.2.2.4 DNA EXTRACTION BY PERFECTPREP GEL CLEANUP 27
2.2.3 RNA PREPARATION 27
2.2.3.1 RNA ISOLATION FROM MAMMALIAN CELLS 27
2.2.4 DNA MODIFICATION 27
2.2.4.1 RESTRICTION ANALYSIS 27
2.2.4.2 DEPHOSPHORYLATION BY ALKALINE PHOSPHATASE
2.2.4.3 GENERATION OF BLUNT ENDS
2.2.4.4 DIMERIZATION OF OLIGONUCLEOTIDES
2.2.4.5 LIGATION OF DNA FRAGMENTS
2.2.4.6 PREPARATION OF COMPETENT BACTERIA
2.2.4.7 SITE-SPECIFIC MUTAGENESIS
2.2.5 TRANSFORMATION
2.2.6 POLYMERASE CHAIN REACTION (PCR)
2.2.7 REAL-TIME PCR
2.2.7.1 REVERSE TRANSCRIPTASE REACTION
2.2.7.2 SEQUENCING OF DNA
2.2.8 BIOCHEMICAL METHODS
2.2.8.1 PREPARATION OF NUCLEAR EXTRACTS AND DETERMINATION OF PROTEIN
CONCENTRATION
2.2.8.2 EIECTROPHORETIC MOBILITY SHIFT ASSAY (L?MSA)
TABLE OF CONTENTS
2.2.9 FUNCTIONAL ANALYSIS 34
2.2.9.1 LUCIFERASE ASSAY 34
3. RESULTS 35
3.1 CONTROVERSIAL TRANSCRIPTIONAL REGULATION
OF MIR-29A IN HSC AND
IN HCC CELLS BY
TGF-PI 35
3.1.1 LOW MIR-29A EXPRESSION IN HEPATIC CARCINOMA CELLS, BUT HIGH
EXPRESSION IN MYOFIBROBLASTS
STELLATE CELLS 35
3.1.2 TGF-PL DOWN-REGULATED MIR-29A EXPRESSION IN HEPATIC STELLATE CELL
LINE 36
3.1.3 EXPRESSION OF MIR-29A WAS INDUCED BY TGF-PI IN HEPATOMA CELL LINE
37
3.2 THE PROMOTER OF THE MIR-29A/BI GENE
CARRIES PUTATIVE BINDING
SITES FOR TGF
-PI
REGULATION 38
3.2.1 IDENTIFICATION OF PUTATIVE REGULATORY BINDING SITES IN THE 5
UPSTREAM REGION OF THE MIR-29A/BL
GENE 39
3.2.2 MIR-29A/BL PROMOTER ACTIVITY IN HSCS AND HEPATOMA CELLS 40
3.2.3 THE CORE-PROMOTER OF MIR-29A/BL WAS IDENTIFIED BY 5 END DELETION
ANALYSIS 41
3.3 TRANSCRIPTIONAL REGULATION OF
MIR-29A/B! BY PHORBOLESTER STIMULATION 43
3.3.1 MIR-29A/BL GENE REGULATION BY NF-KB IN HEPATOMA, BUT NOT IN
STELLATE CELLS 43
3.3.2 PRONOUNCED INDUCTION OF MIR-29 EXPRESSION IN HEPATOMA CELLS BY
PHORBOLESTER STIMULATION ...45
3.4 MIR-29A/B! PROMOTER REGULATION BY TGF-P
IN HSCS 46
3.4.1 IDENTIFICATION OFTGF-P RESPONSIVE ELEMENTS IN THE MIR-29A/BL
REGULATORY REGION 46
3.4.2 TGF-P REDUCES MIR-29A EXPRESSION VIA A SMAD BINDING SITE IN THE
MIR-29A/B! CORE-PROMOTER
50
3.5 THE MEMBERS
OF THE API
TRANSCRIPTION FACTOR FAMILY
PROMINENTLY CONTRIBUTE
TO
TGF-P STIMULATED
MIR-29A/BI GENE INDUCTION
IN HEPATOMA
CELLS 53
3.5.1 STIMULATION OF MIR-29A/BL PROMOTER ACTIVITY BY TGF-PL DEPENDS ON
THE APIA SITE AND ITS
INTERACTION WITH HETEROGENOUS TRANSCRIPTION COMPLEXES 53
3.5.2 API FAMILY IS A PROMINENT ACTIVATING TRANSCRIPTION FACTOR FOR
MIR-29 INDUCTION IN HEPATOMA
CELLS IN RESPONSE TO TGF-P! AND PHORBOLESTER 56
TABLE OF CONTENTS
4. DISCUSSION 62
4.1 TGF-P MEDIATES
MIR-29A REPRESSION IN
HSC 62
4.1.1 REGULATION OF MIR-29A/BL PROMOTER ACTIVITY IN HSC BY TGF-P 63
4.1.2 BINDING SITES ON THE MIR-29A/B I PROMOTER INVOLVED IN TGF-P
REGULATION IN HSC 64
4.1.3 TGF-P REDUCES MIR-29A EXPRESSION BY A SMAD BINDING SITE IN HSC 65
4.2 TGF-P DECREASES
MIR-29A EXPRESSION IN
HEPATOMA CELLS 66
4.2.1 TGF-P REGULATES INIR-29A/BL PROMOTER ACTIVITY IN HEPATOMA CELLS 67
4.2.2 TRANSCRIPTION OF MIR-29A/BL IS REGULATED BY TGF-P I THROUGH APIA
SITE 68
4.2.3 COMPETITOR-CENTERED INTERACTION AT THE APIA REGION HARBOURING A
APL AND AN ET-I SITE 68
5. SUMMARY 71
S.I ZUSAMMENFASSUNG 73
6. REFERENCES 75
7. LEBENSLAUF 83
|
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| ctrlnum | (OCoLC)903693652 (DE-599)DNB1065415214 |
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| dewey-ones | 610 - Medicine and health |
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| discipline | Medizin |
| format | Thesis Book |
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| language | English |
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| physical | 84 S. Ill., graph. Darst, 21 cm |
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| spelling | Huang, Jia 1984- Verfasser (DE-588)1064989969 aut Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta vorgelegt von Jia Huang 2014 84 S. Ill., graph. Darst, 21 cm txt rdacontent n rdamedia nc rdacarrier Köln, Univ., Diss., 2014 (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=027833100&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Huang, Jia 1984- Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta |
| subject_GND | (DE-588)4113937-9 |
| title | Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta |
| title_auth | Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta |
| title_exact_search | Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta |
| title_full | Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta vorgelegt von Jia Huang |
| title_fullStr | Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta vorgelegt von Jia Huang |
| title_full_unstemmed | Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta vorgelegt von Jia Huang |
| title_short | Regulation of the microRNA-29a/b1 gene in liver cells by transforming growth factor beta |
| title_sort | regulation of the microrna 29a b1 gene in liver cells by transforming growth factor beta |
| topic_facet | Hochschulschrift |
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