MicroRNAs in malignant tumors of the skin: First steps of tiny players in the skin to a new world of genomic medicine

251 Pages·2016·9.022 MB·English

by  PD Dr. Michael Sand (auth.)

Michael Sand
MicroRNAs in
malignant tumors
of the skin
First steps of tiny players in the skin
to a new world of genomic medicine
MicroRNAs in malignant tumors of the skin

Michael Sand
MicroRNAs in malignant
tumors of the skin
First steps of tiny players in the skin to
a new world of genomic medicine
Priv.-Doz. Dr.med. Michael Sand
Bochum, Germany
ISBN 978-3-658-12793-0 ISBN 978-3-658-12794-7 (eBook)
DOI 10.1007/978-3-658-12794-7

Library of Congress Control Number: 2016939488

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Acknowledgments
I would first and foremost like to thank my clinical and scientific teacher,
Professor Peter Altmeyer, for his support far above and beyond the
norm, his excellent loyalty, and the unlimited access to all the needed
resources of the clinic, which has fostered my personal and academic
development to a great degree.
Special thanks goes to the Head of Dermatological Surgery,
Professor Falk G. Bechara, for his unparalleled support in the
development of scientific questioning and ideas, as well as for the
education on performing operations, which I was able to gain in his
department. As an academic teacher and as a friend, he has enabled the
creation of this work with his extraordinary backing.
I would like to deeply thank Dr. Marina Skrygan, who stood by my
side in cases of molecular-biological questions with impressive specialist
knowledge under great personal involvement over the past years, and
through this has contributed massively to the creation of this work.
I would like to thank my colleague Mr. Dimitrios Georgas for the
excellent sample coordination in the Dermatological Surgery unit, and
the Director of the Skin Tumor Center, Professor Thilo Gambichler for
the extraordinary expert knowledge in statistical questioning and in the
development of scientific questioning.
I would especially like to express my particular thanks to my mother
and my brother, who have enabled this work through their patience and
their encouragement in the past years.

Table of Contents
1 Introduction ...................................................................................... 1
1.1 MicroRNAs .............................................................................. 1
1.2 MicroRNA maturity and function .............................................. 2
1.3 MicroRNA in oncology ............................................................. 5
1.4 MicroRNA in skin tumors ......................................................... 7
2 Objectives of this work ................................................................. 15
3 Summary and discussion of this work ........................................ 17
3.1 Studies on non-melanocytic, epithelial skin tumors .............. 17
3.1.1 Expression of the miRNA maturity enzymes ............. 17
3.1.2 Expression of DGCR8 and the RISC components .... 21
3.1.3 Expression profiles of microRNAs in basal cell
carcinoma .................................................................. 28
3.1.4 miRNA expression profiles in cutaneous squamous
cell carcinomas .......................................................... 30
3.2 Examinations on malignant melanoma ................................. 36
3.2.1 Expression of the miRNA maturity enzymes,
transporters, and RISC components ......................... 36
3.2.2 Immunohistochemical study of the miRNA maturity .. 44
3.2.3 miRNA expression profiles in melanoma .................. 47
4 Closing remarks ............................................................................. 53
5 Paper 1: Expression levels of the microRNA processing
enzymes Drosha and Dicer in epithelial skin cancer ................. 55
5.1 Introduction ............................................................................ 55
5.2 Materials and Methods .......................................................... 56
5.2.1 Subjects ..................................................................... 56
5.2.2 RNA isolation and real-time RT-PCR ........................ 56
5.2.3 Statistical analyses .................................................... 57
5.3 Results ................................................................................... 58
5.4 Discussion ............................................................................. 60
5.5 Conclusion ............................................................................. 64
5.6 References ............................................................................ 64
VIII Table of Contents
6 Paper 2: Expression levels of the microRNA maturing
microprocessor complex component DGCR8 and the RNA-
induced silencing complex (RISC) components argonaute-1,
argonaute-2, PACT, TARBP1 and TARBP2 in epithelial skin
cancer ............................................................................................. 67
6.1 Introduction ............................................................................ 67
6.2 Materials and Methods .......................................................... 68
6.3 Results ................................................................................... 70
6.4 Discussion ............................................................................. 71
6.5 Conclusion ............................................................................. 77
6.6 References ............................................................................ 77
7 Paper 3: Expression of microRNAs in basal cell carcinoma .... 81
7.1 Introduction ............................................................................ 81
7.2 Materials and Methods .......................................................... 81
7.2.1 Subjects ..................................................................... 82
7.2.2 RNA isolation ............................................................. 82
7.2.3 Preparation of labeled miRNA, microarray
hybridization and scanning ........................................ 82
7.2.4 TaqMan quantitative real-time reverse transcription
polymerase chain reaction (qRT-PCR) ..................... 84
7.2.5 Bioinformatic data analyses....................................... 85
7.2.6 Statistical analyses .................................................... 85
7.2.7 Data mining analysis ................................................. 86
7.3 Results ................................................................................... 86
7.3.1 Quality control ............................................................ 86
7.3.2 Differential miRNA expression based on
microarray data .......................................................... 86
7.3.3 Correlation analysis ................................................... 87
7.3.4 Cluster analysis ......................................................... 87
7.3.5 TaqMan qRT-PCR validation..................................... 88
7.3.6 Data mining results .................................................... 88
7.4 Discussion ............................................................................. 88
7.5 Conclusion ............................................................................. 96
7.6 Acknowledgements ............................................................... 97
7.7 References ............................................................................ 97
Table of Contents IX
8 Paper 4: Microarray analysis of microRNA expression in
cutaneous squamous cell carcinoma ........................................ 101
8.1 Introduction .......................................................................... 101
8.2 Materials and Methods ........................................................ 102
8.2.1 RNA isolation ........................................................... 102
8.2.2 Preparation of labeled miRNA, microarray
hybridization and scanning ...................................... 102
8.2.3 TaqMan quantitative real-time reverse transcription
polymerase chain reaction (qRT-PCR) ................... 103
8.2.4 Bioinformatic Data analyses .................................... 104
8.2.5 Statistical analyses .................................................. 107
8.2.6 Mining Data Analysis ............................................... 107
8.3 Results ................................................................................. 109
8.3.1 Quality control .......................................................... 109
8.3.2 Differential miRNA expression ................................ 109
8.3.3 Correlation analysis ................................................. 109
8.3.4 Cluster analysis ....................................................... 110
8.3.5 TaqMan RT-PCR Validation .................................... 112
8.3.6 Mining data analysis results .................................... 113
8.4 Discussion ........................................................................... 113
8.5 Acknowledgements ............................................................. 120
8.6 References .......................................................................... 120
9 Paper 5: The miRNA machinery in primary cutaneous
malignant melanoma, cutaneous malignant melanoma
metastases and benign melanocytic nevi ................................. 127
9.1 Introduction .......................................................................... 127
9.2 Materials and Methods ........................................................ 129
9.2.1 Subjects ................................................................... 129
9.2.2 RNA isolation and TaqMan real-time reverse
transcription polymerase chain reaction (RT-PCR) . 129
9.2.3 Statistical analyses .................................................. 131
9.3 Results ................................................................................. 135
9.4 Discussion ........................................................................... 136
9.5 Conclusion ........................................................................... 148
9.6 Acknowledgements ............................................................. 148
9.7 References .......................................................................... 148