Analysis of the miRNA-mediated regulation of AP-1 in non-tumorigenic and tumorigenic HPV18-positive cell lines [Elektronische Ressource] / vorgelegt von Matthias Sobel

Analysis of the miRNA-mediated regulation of AP-1 in
non-tumorigenic and tumorigenic HPV18-positive cell lines





INAUGURAL-DISSERTATION
zur
Erlangung der Doktorwürde
der
Naturwissenschaftlich-Mathematischen Gesamtfakultät
der
Ruprecht-Karls-Universität
Heidelberg




vorgelegt von
Apotheker
Matthias Sobel
geb. in Mainz



November 2009

Analysis of the miRNA-mediated regulation of AP-1 in
non-tumorigenic and tumorigenic HPV18-positive cell lines









Die vorliegende Arbeit entstand im Zeitraum von Oktober 2006 to Oktober 2009 am Deut-
schen Krebsforschungszentrum (DKFZ) Heidelberg unter Anleitung von Prof. Dr. Frank Rösl.



Gutachter: Prof. Dr. Elisabeth Schwarz
Deutsches Krebsforschungszentrum Heidelberg


Prof. Dr. Frank Rösl
Deutsches Krebsforschungszentrum Heidelberg


Tag der mündlichen Prüfung:


















Eidesstattliche Versicherung
Ich erkläre hiermit, dass ich die vorgelegte Dissertation selbst verfasst und mich dabei keiner
anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient habe. Diese
Dissertation wurde in dieser oder in anderer Form weder bereits als Prüfungsarbeit verwen-
det, noch einer anderen Fakultät als Dissertation vorgelegt. An keiner anderen Stelle ist ein
Prüfungsverfahren beantragt.

Heidelberg, den 18.11.2009


Matthias Sobel







für meine Eltern











Table of contents 5
Table of contents
Table of contents………………………………………………………………………………………5
Summary……………………………………………………………………………………………...10
Zusammenfassung…………………………………………………………………………………..11
1. Introduction ...................................................................................................................12
1.1. Cervical cancer and human papillomaviruses..................................................12
1.1.1. Genomic organization and replication of HPV ...............................................13
1.1.2. Viral oncogenes E6 and E7...........................................................................14
1.2. The transcription factor AP-1.............................................................................15
1.2.1. Composition of the transcription factor AP-1 .................................................15
1.2.2. AP-1 family members: characteristics, function and regulation......................16
1.2.3. Role of AP-1 in HPV......................................................................................18
1.3. microRNAs ..........................................................................................................20
1.3.1. Biogenesis of microRNAs..............................................................................20
1.3.2. Repression mechanisms of miRNAs.……………………….………..…………..22
1.3.3. microRNAs and cancer .................................................................................23
1.3.4. microRNAs and cervical cancer.....................................................................25
1.3.5. microRNAs and AP-1 ....................................................................................26
1.4. Aim of this study.................................................................................................27
2. Material .........................................................................................................................28
2.1. Chemicals and reagents.....................................................................................28
2.2. Reagents and media for bacteria culture ..........................................................30
2.3. Reagents and media for cell culture..................................................................30
2.4. Oligonucleotides.................................................................................................31
2.4.1. siRNAs and miRIDIAN® miRNA mimics for knock-down experiments...........31
2.4.2. double-stranded oligonucleotides for gene analysis by EMSA.......................31
2.4.3. Single-stranded oligonucleotides as Northern probes for miRNA analysis.....32
2.4.4. Oligonucleotides for protein-coding gene analysis by RT-PCR......................32 Table of contents 6
2.4.5. Oligonucleotides for miRNA analysis by RT-PCR..........................................33
2.4.6. Oligonucleotides for 3’UTR cloning by RT-PCR ............................................33
2.4.7. Oligonucleotides for sequencing....................................................................35
2.5. Plasmids..............................................................................................................35
2.5.1. Original plasmids...........................................................................................35
2.5.2. Modified plasmids .........................................................................................35
2.6. Enzymes ..............................................................................................................36
2.7. Size Markers........................................................................................................37
2.8. Kits.......................................................................................................................37
2.9. Antibodies ...........................................................................................................37
2.10. Consumables ......................................................................................................39
2.11. Apparatuses & laboratory equipment ...............................................................40
2.12. Cell lines..............................................................................................................41
2.13. Solutions and buffers .........................................................................................42
2.14. Search engines ...................................................................................................47
3. Methods ........................................................................................................................49
3.1. Cultivation of eukaryotic cells ...........................................................................49
3.1.1. Cell culture ....................................................................................................49
3.1.2. Cryoconservation and reactivation of eukaryotic cells ...................................49
3.1.3. Cell counting .................................................................................................49
3.2. Preparation and analysis of proteins ................................................................50
3.2.1. Nuclear protein preparation (Schreiber et al., 1989) ......................................50
3.2.2. Whole cell protein preparation (RIPA) (Klotz et al., 1999)..............................50
3.2.3. Protein quantification according to Bradford (Bradford, 1976) .......................51
3.2.4. Western blot analysis ....................................................................................51
3.2.5. Electrophoresis mobility shift assay (EMSA)..................................................53
3.3. Preparation and analysis of nucleic acids ........................................................54
3.3.1. Genomic DNA preparation ............................................................................54
3.3.2. Cytoplasmic RNA preparation .......................................................................55 Table of contents 7
3.3.3. Total RNA preparation...................................................................................55
3.3.4. Nucleic acid quantification.............................................................................56
3.3.5. RNA agarose gel electrophoresis..................................................................56
3.3.6. Reverse transcription ....................................................................................57
3.3.7. Semi-quantitative polymerase chain reaction ................................................57
3.3.8. Quantitative polymerase chain reaction.........................................................58
3.3.9. Northern blot analysis....................................................................................59
3.4. Cloning techniques: preparation of reporter plasmids ....................................61
3.4.1. Cloning strategies .........................................................................................62
3.4.2. RNase H digestion ........................................................................................63
3.4.3. Semi-quantitative PCR to amplify 3’UTRs for cloning ....................................63
3.4.4. Analytical and preparative restriction enzyme digestion ................................64
3.4.5. Purification of DNA................................................................................