Identification of microRNAs miR-203 and miR-335 forming a network of regulation in breast cancer development [Elektronische Ressource] / Holger Heyn

Holger Heyn

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2010
• Nombre de lectures: 30
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Identification of microRNAs miR-203 and miR-335 forming a network of
regulation in breast cancer development




Von der naturwissenschaftlichen Fakultät
der Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades
DOKTOR DER NATURWISSENSCHAFTEN
Dr. rer. nat.



genehmigte Dissertation
von

Diplom-Biologe Holger Heyn

Geboren am 29.01.1979,
in Bremen

2010

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Referentin: Prof. Dr. Schlegelberger

Korreferentin: Prof. Dr. Gerardy-Schahn


Tag der Promotion:18.12.2009



Schlagworte: MikroRNA, BRCA1, Brustkrebs

Keywords: MicroRNA, BRCA1, Breast cancer
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Content
Content


1 Summary ...................................................................................................................... 1

1.1 Zusammenfassung ................................... 2


2 Introduction ................................................................................................................. 3

2.1 BRCA1 and breast cancer formation ....... 3

2.2 The inhibitor of DNA binding (ID4) ....................................................................... 5

2.3 The estrogen receptor α (ERα) ................ 6

2.4 The aryl hydrocarbon receptor (AhR) ..................................................................... 7

2.5 The insulin-like growth factor 1 receptor (IGF1R) ................. 8

2.6 The specificity protein 1 (SP1) ................................................................................ 9

2.7 The tightly cross-linked network of BRCA1 ......................... 10

2.8 MicroRNAs and their function .............................................................................. 11

2.9 Control of microRNA expression and biogenesis ................. 15

2.10 MicroRNAs involved in cancer ............................................................................. 16

2.11 MicroRNAs as diagnostic tools and therapeutic targets ....... 18


3 Aim of the study ......................................................................................................... 21


4 Material and Methods ............................................................................................... 22

4.1 Cell culture and modification ................................................................................ 22

4.1.1 Cell culture .................................... 22
4.1.2 5-Aza treatment ............................................................. 23
4.1.3 Estradiol stimulation ...................................................................................... 23
4.1.4 Oligonucleotides and plasmids ...................................... 23

4.1.4.1 microRNAs............................. 23
4.1.4.2 siRNAs ................................................................... 24
4.1.4.3 Plasmids ................................. 24
4.1.4.4 Co-transfections ..................................................................................... 26
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Content

4.2 Patient material ...................................................................................................... 27

4.2.1 Clinical features ............................. 27

4.3 MicroRNA co-precipitation .................................................................................. 27

4.3.1 In vitro transcription ...................... 27
4.3.2 mRNA transfection ........................ 28
4.3.3 Co-precipitation ............................................................................................. 28

4.4 Expression analysis ............................... 29

4.4.1 Microdissection ............................................................................................. 29
4.4.2 RNA isolation ................................ 30
4.4.3 Reverse transcription ..................... 30
4.4.4 Quantitative real-time PCR (qRT-PCR) ........................ 30
4.4.5 Protein isolation ............................................................................................. 31
4.4.6 Western blotting ............................ 32
4.4.7 Isolation of genomic DNA and MSP 32

4.5 Functional analysis ................................................................................................ 33

4.5.1 Viability assay ............................... 33
4.5.2 Apoptotic assay ............................. 33
4.5.3 Cell cycle analysis ......................................................................................... 34
4.5.4 Luciferase assay 34

4.6 Statistics................................................. 34


5 Results ......................................................................................................................... 36

5.1 MicroRNAs control the regulatory cascade of BRCA1 ......................................... 36

5.2 MicroRNA co-precipitation enabled the detection of predicted ..............................
mic:mRNA interactions ............................................. 40

5.2.1 MicroRNA Let-7 co-immunoprecipitated with NRAS .................................. 42
5.2.2 Mic miR-335 bound to ID4 mRNA ................................................... 45

5.3 Reporter assays demonstrated direct microRNA:target interaction ...................... 47

5.4 The microRNAs miR-203 and miR-335 influenced the expression of BRCA1 .... 48

5.5 MiR-203 and miR-335 influenced cellular behavior and fate ............................... 49

5.5.1 MiR-203 induced apoptosis and decelerated growth .................................... 49
5.5.2 MiR-335 induced ais in cancer cells ................... 51

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Content
5.6 MicroRNA miR-203 and miR-335 expressions were altered in human ..................
sporadic breast cancer ........................................................................................... 53

5.7 ID4 revealed a crucial function in the microRNA-dependent network................. 56

5.8 The promoter regions of miR-203 and miR-335 harbored different .........................
regulatory elements ............................................................................................... 59

5.8.1 The transcription factor SP1 regulated the expression of miR-203............... 59
5.8.2 The expression of miR-203 and miR-335 was induced by estrogen ............. 65


6 Discussion ................................................................................................................... 67


7 Future perspectives ................................................................................................... 87


8 References................................................................................................................... 89


9 List of abbreviations ................................................................................................ 105

9.1 List of abbreviations (chemicals) ........ 106

9.2 List of abbreviations (genes and proteins) .......................................................... 106


10 Erklärung zur Dissertation ..................................................... 108


11 Curriculum Vitae ..................................................................................................... 109


12 Danksagungen .......... 110

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Summary
1 Summary

Human breast cancer, representing the most frequent tumor in women, can be divided into
two major subclasses, the inherited and the sporadic form. Whereas the inherited subclass
is predominantly characterized by mutations of the cancer susceptibility genes BRCA1 and
BRCA2, the underlying mechanisms leading to sporadic breast cancer remain undefined
and are therefore subject of the present study.
Here, we focus on the decipherment of the complex network regulating BRCA1, involving
the transcriptional activators ERα, IGF1R, AhR and SP1, as well as the dominant negative
repressor ID4. Deregulation of individual or multiple components of this network promote
breast cancer formation by repressing BRCA1 as a key molecule for genomic stability and
by activating mitogenic signal cascades.
In this study, we analyzed post-transcriptional control mechanisms mediated by
microRNAs, which could previously be identified as regulator of crucial cellular functions.
We investigated the role of two independent microRNAs, miR-203 and miR-335, for the
formation of sporadic human breast cancer and their involvement in the regulatory network
of the cancer susceptibility gene BRCA1.
MiR-335 was found at a decreased expression level in primary sporadic breast cancer
specimens, positively correlating to the transcript level of BRCA1. Functionally,
overexpression of miR-335 led to decreased cell viability, paralleled by an increase in
apoptosis and downregulation of the BRCA1 activators ERα, SP1, AhR and IGF1R and the
repressor ID4, suggesting a tumor-suppressive fun