Identification of a DNA methylation signature and distinct microRNA variants in breast cancer [Elektronische Ressource] / presented by Yasser Riazalhosseini

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Inaugural Dissertation Submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Master Sci. Yasser Riazalhosseini Born in Tehran, Iran Oral-examination: June 17, 2010 Identification of a DNA methylation signature and distinct microRNA variants in breast cancer This work was carried out in the Division of Functional Genome Analysis at the German Cancer Research Center (DKFZ) Head of division: Dr. Jörg Hoheisel Referees: Prof. Dr. Harald Hermmann-Lerdon Prof. Dr. Jörn WalterThesis declaration I hereby certify that this thesis was written by me. No sources of information other than those listed in the references were used. Heidelberg, April 27, 2010 …………………………… Yasser Riazalhosseini iii Acknowledgement I am grateful to; - My parents who were the first teachers in my life and Maria Shahmoradgoli for her constant support throughout our life. - Jörg Hoheisel for giving me the opportunity to carry out this research towards my doctorate, his invaluable ideas and support, and the unique friendly atmosphere of his group. - Verena Beier for the introduction to the microarray field and technical guidance. - Prof. Dr. Harald Hermmann-Lerdon (DKFZ) and Prof. Dr.

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Inaugural Dissertation
Submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences







Presented by
Master Sci. Yasser Riazalhosseini
Born in Tehran, Iran
Oral-examination: June 17, 2010


Identification of a DNA methylation signature and
distinct microRNA variants in breast cancer


This work was carried out in the Division of Functional Genome Analysis
at the German Cancer Research Center (DKFZ)
Head of division: Dr. Jörg Hoheisel






Referees:
Prof. Dr. Harald Hermmann-Lerdon
Prof. Dr. Jörn WalterThesis declaration
I hereby certify that this thesis was written by me. No sources of information other than
those listed in the references were used.


Heidelberg, April 27, 2010


……………………………
Yasser Riazalhosseini
iii
Acknowledgement
I am grateful to;
- My parents who were the first teachers in my life and Maria Shahmoradgoli for her
constant support throughout our life.
- Jörg Hoheisel for giving me the opportunity to carry out this research towards my
doctorate, his invaluable ideas and support, and the unique friendly atmosphere of
his group.
- Verena Beier for the introduction to the microarray field and technical guidance.
- Prof. Dr. Harald Hermmann-Lerdon (DKFZ) and Prof. Dr. Jörn Walter (Universität
des Saarlandes), members of the advisory committee of my PhD thesis, for their
insightful advice.
- Sandeep Kumar Botla and Pedro de Souza Rocha Simonini for their tremendous
help in the laboratory work and also in the preparation of the graphical images.
- Amin Moghaddas Gholami (DKFZ) for his broad and valuable bioinformatic
analysis.
- Dr. Achim Breiling and Prof. Dr. Frank Lyko (DKFZ) for the ChIP analysis and our
fruitful collaboration.
- Michaela Schanne for the translation of the Summary of my thesis from English to
German.
- Evgenij Moskalev, Neeme Tõnisson, Michael Böttcher, Armin Pscherer, Özgür
Sahin and Doris Mayer for helpful discussions.
- Achim Stephan, Marita Schrenk, Azin Jahangiri for their technical support.
- Christoph Schröder, Jorge Soza Ried and Mahmoud Mostafa Youns for their
support and good friendship.
- All my former and current colleagues in the Division of Functional Genome
Analysis and my friends at the DKFZ.
iv
Table of Contents
Thesis declaration .................................................................................................................. iii
Acknowledgement ................. iv
Table of Contents ................................................................................................................... v
Abbreviations ........................ ix
Summary ................................................................................................................................ xi
Zusammenfassung ............................................................................................................... xiii
1 Introduction .................... 1
1.1 Cancer ..................................................................................................................... 1
1.1.1 Breast Cancer .................................................................................................... 2
1.2 Epigenetics ............. 4
1.2.1 DNA methylation ............................................................................................. 5
1.2.2 Covalent histone modifications ........................................ 5
1.2.3 microRNAs (miRNAs) ..................................................... 7
1.3 Epi-Genetics of Cancer ........................................................... 7
1.3.1 Abnormal DNA methylation patterns in cancer ............................................... 9
1.3.2 miRNAs and cancer ........................................................ 14
1.3.3 Epi-Genetics of breast cancer ......................................................................... 14
1.3.4 Aim ................................................. 17
2 Material and Methods ................................................................................................... 18
2.1 Materials ............................................................................................................... 18
2.1.1 Instruments ..... 18
v
2.1.2 Chemical reagents, enzymes and general materials ....................................... 19
2.1.3 Cell culture ..................................................................... 21
2.1.4 Kits ................................................. 21
2.1.5 Media, solutions and buffers .......................................... 22
2.1.6 Antibodies ....................................................................... 24
2.1.7 Plasmids and synthetic RNAs ........................................ 24
2.1.8 qRT-PCR primers ........................................................... 25
2.1.9 Genomic primers for the amplification of CGIs from genomic DNA ............ 25
2.1.10 SoBi primers for the amplification of CGIs from deaminated DNA .............. 27
2.1.11 Primers used for ChIP analysis of miR-375 locus .......................................... 28
2.1.12 Primers used for isolation of miR-375 target sites ......... 29
2.1.13 Cell lines ......................................................................................................... 29
2.2 Methods ................ 29
2.2.1 General molecular biology methods ............................................................... 29
2.2.2 Processing of tissue samples for DNA/RNA isolation ................................... 37
2.2.3 Selection of CGIs and designing PCR primers to isolate them from
deaminated DNA .......................................................................................................... 38
2.2.4 Preparation of the control samples . 40
2.2.5 Sodium bisulfite treatment of DNA samples .................................................. 43
2.2.6 Isolation of selected CGIs from patients DNA ............... 45
2.2.7 Labeling of DNA by random priming ............................................................ 46
2.2.8 Microarrays for DNA methylation analysis ................... 48
2.2.9 Data analysis ................................................................................................... 54
2.2.10 Bisulfite-sequencing ....................... 56
2.2.11 Cell culture ..................................................................................................... 57
vi
2.2.12 Chromatin Immunoprecipitation (ChIP) assay ............................................... 59
2.2.13 Expression arrays ............................................................ 59
2.2.14 Functional analysis ......................................................... 62
2.2.15 Statistical analysis .......................................................... 66
3 Results ................................................................ 67
3.1 Microarray based analysis of DNA methylation patterns .................................... 67
3.1.1 Developing microarrays for DNA methylation analysis 67
3.1.2 Optimization of probe length for reliable array performance ........................ 69
3.1.3 Selection of highly discriminative probes ...................................................... 75
3.2 Screening DNA methylation patterns in tissue samples ....................................... 75
3.2.1 Isolation of DNA from tissue samples ........................................................... 75
3.2.2 PCR-amplification of CGIs from DNA samples ............................................ 76
3.2.3 Microarray-based analysis of methylation patterns in tissue samples ............ 77
3.2.4 Bisulfite sequencing data ................................................................................ 85
3.2.5 Re-activation of methylated genes following decitabine treatment ............... 89
3.3 Identification of miRNAs involved in ERα activation ......................................... 90
3.3.1 miR-375 expression is increased in ERα-positive breast cancer cell lines .... 90
3.3.2 Reciprocal regulation between miR-375 and ERα ......................................... 92
3.3.3 miR-375 inhibition reduces the proliferation rate of breast cancer cells ........ 93
3.3.4 Epigenetic marks determine the transcriptional state of the miR-375 locus .. 94
3.3.5 Transcriptional repressors bind to the miR-375 locus .................................... 95
3.3.6 RASD1 is a functional target of miR-375 ...................................................... 99
3.3.7 Regulatory connection between ERα and RASD1 ....... 102
4 Discussion ................................................................................................................... 104
vii
4.1 DNA methylation analysis ................................................................................. 104
4.1.1 Development of microarrays ........ 104
4.1.2 DNA methylation patterns of breast tumors ................................................. 107
4.1.3 Genes affected by hypermethylation in tumors ............ 108
4.1.4 DNA methylator event in breast cancer? ...................................................... 112
4.1.5 DNA methylation signature for diagnosis of breast cancer .......................... 114
4.1.6 DNA methylation patterns at signature loci ................................................. 116
4.2 Identification of miRNAs involved in ERα activation ....... 118
4.2.1 Epigenetic marks at miR-375 locus .............................................................. 119
4.2.2 Proposed model for RASD1 and miR-375 effects on ERα .......................... 121
5 Outlook ....................................................................................................................... 123
6 References .................. 125
7 Appendix .................................................................................................................... 136
7.1 Supplementary Table S1 ..................... 136
7.2 Supplementary Table S2 ................................................................ 137
7.3 Supplementary Table S3 ..................... 137
7.4 Supplementary Table S4 ................................................................ 138
7.5 Supplementary Table S5 ..................... 138
7.6 Publications ........................................................................................................ 142


viii
Abbreviations
ATP Adenosine triphosphate
BSA Bovine serum albumin
bp Base pair
cDNA Complementary DNA
CCD Charge-Coupled Device
CGI CpG island
ChIP Chromatin immunoprecipitation
dNTP Desoxynucleotide triphosphate
DNA Desoxyribonucleic acid
DNMT DNA methyltransferase
dUTP Desoxyuridine triphosphate
ERα Estrogen receptor α
ERE Estrogen responsive element
E. coli Escherichia coli
FDR False discovery rate
kb Kilobase
miRNA microRNA
mRNA Messenger RNA
PAGE Polyacrylamide gel electrophoresis
PCR Polymerase chain reaction
PVDF Polyvinylidene fluoride
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