The Role of microRNA in Myogenesis and Rhabdomyosarcoma

149 Pages·2014·3 MB·English

by  A. Taylor Bright

UC San Diego
UC San Diego Electronic Theses and Dissertations
Title
The Role of microRNA in Myogenesis and Rhabdomyosarcoma
Permalink
https://escholarship.org/uc/item/4hh379v9
Author
Muir, Shannon E.
Publication Date
2014

Peer reviewed|Thesis/dissertation
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University of California
UNIVERSITY OF CALIFORNIA, SAN DIEGO

The Role of microRNA in Myogenesis and Rhabdomyosarcoma

A dissertation submitted in partial satisfaction of the
requirements for the degree Doctor of Philosophy

in

Biomedical Sciences

by

Shannon E. Muir

Committee in charge:

Professor Karen Arden Chair
Professor Web Cavenee, Co-Chair
Professor Frank Furnari
Professor Xiang-Dong Fu
Professor Amy Pasquinelli
Professor Eugene Yeo

2014
©

Shannon E. Muir, 2014

All rights reserved.
SIGNATURE PAGE

The Dissertation of Shannon E. Muir is approved, and it is acceptable in
quality and form for publication on microfilm and electronically:

Co- Chair
Chair

University of California, San Diego

2014
iii
DEDICATION

For Jacom Derick Larson
August 28th, 1978 - October 21st, 2008

iv
TABLE OF CONTENTS
SIGNATURE PAGE ............................................................................................................... iii

DEDICATION ......................................................................................................................... iv

TABLE OF CONTENTS ......................................................................................................... v

LIST OF FIGURES ................................................................................................................. ix

LIST OF TABLES .................................................................................................................... x

ACKNOWLEDGEMENTS .................................................................................................... xi

VITA ........................................................................................................................................ xii

ABSTRACT OF THE DISSERTATION ............................................................................ xvii

CHAPTER 1- INTRODUCTION ............................................................................................ 1
MYOGENESIS ............................................................................................................. 1
RHABDOMYOSARCOMA ............................................................................................. 3
Overview .............................................................................................................................................. 3
Treatment ........................................................................................................................................... 4
Associated Syndromes ................................................................................................................... 5
Diagnosis ............................................................................................................................................. 6
THE FUSION PROTEIN ................................................................................................ 8
PAX3 ...................................................................................................................................................... 8
FOXO1a .............................................................................................................................................. 10
PAX3-FOXO1a ................................................................................................................................. 11
Pediatric vs. adult rhabdomyosarcoma ............................................................................. 14
MICRORNA ............................................................................................................. 14
MicroRNA in Myogenesis .......................................................................................................... 16

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MicroRNA in Cancer .................................................................................................................... 18
MicroRNA in Rhabdomyosarcoma ....................................................................................... 19
MicroRNA-495 ............................................................................................................................... 22
HIGH-THROUGHPUT SEQUENCING .......................................................................... 24
small RNA-seq ................................................................................................................................ 26
SUMMARY ................................................................................................................ 26
FIGURES ................................................................................................................... 27

CHAPTER 2 - SKELETAL MUSCLE CELLS HAVE DIFFERENT MICRORNA
TRANSCRIPTOMES THAN THOSE OF RHABODOMYOSARCOMA CELLS ......... 29
INTRODUCTION ........................................................................................................ 29
RESULTS .................................................................................................................. 31
Small RNA-seq identifies miRNAs known to be differentially expressed between
differentiated and undifferentiated primary skeletal muscle cells ...................... 31
Differentiated and undifferentiated skeletal muscle cells have unique miRNA
signatures ....................................................................................................................................... 32
Normal skeletal muscle cells and rhabdomyosarcoma cells reflect differential
miRNA expression ....................................................................................................................... 33
ERMS cells and ARMS cells have unique miRNA transcriptomes ........................... 37
DISCUSSION ............................................................................................................. 37
METHODS ................................................................................................................. 40
Cell culture of primary cells and cell lines ........................................................................ 40
Generation of differentiation primary skeletal muscle cells .................................... 40
Western blot analysis ................................................................................................................. 40
RNA harvesting and extraction, and sample preparation ........................................ 41

vi
High throughput sequencing .................................................................................................. 41
Processing small RNA-seq data .............................................................................................. 42
Analysis of small RNA-seq data .............................................................................................. 42
Reverse Transcription PCR and Quantitative Real-Time PCR ................................. 42
Hierarchical clustering .............................................................................................................. 43
ACKNOWLEDGEMENTS ............................................................................................ 44
FIGURES ................................................................................................................... 45
TABLES .................................................................................................................... 54

CHAPTER 3 - PAX3-FOXO1A ALTERS MICRORNA TRANSCRIPTOMES OF
RHABDOMYOSARCOMA CELL LINES .......................................................................... 72
INTRODUCTION ........................................................................................................ 72
RESULTS .................................................................................................................. 74
MiRNA signatures are changed by introduction or knockdown of PAX3-FOXO1a
............................................................................................................................................................. 74
PAX3-FOXO1A expression upregulates three miRNAs ................................................ 74
miR-495 and miR-543 genes are located in non-codingRNA cluster DLK1-DIO3
and have predicted PAX3 binding sites ............................................................................. 75
DISCUSSION ............................................................................................................. 76
METHODS ................................................................................................................. 79
Generation of stable cell lines ................................................................................................. 79
siRNA knockdown of PAX3-FOXO1A .................................................................................... 80
Western blot analysis ................................................................................................................. 80
RNA harvesting and extraction, and sample preparation ........................................ 81
High throughput sequencing .................................................................................................. 81

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Processing small RNA-seq data .............................................................................................. 82
Analysis of small RNA-seq data .............................................................................................. 82
Reverse Transcription PCR and Quantitative Real-Time PCR ................................. 82
Determination of PAX3 binding sites .................................................................................. 83
Generation of PAX3-FOXO1a truncations ......................................................................... 83
ACKNOWLEDGEMENTS ............................................................................................ 84
FIGURES ................................................................................................................... 85
TABLES .................................................................................................................... 90

CHAPTER 4 - MANIPULATION OF MICRORNAS ........................................................ 94
INTRODUCTION ........................................................................................................ 94
RESULTS .................................................................................................................. 95
Inhibition of miRNA-543 increases miR-495 ................................................................... 95
Effect of miRNA inhibition on migration ........................................................................... 95
METHODS ................................................................................................................. 96
Knockdown of miRNAs ............................................................................................................... 96
RNA analysis ................................................................................................................................... 96
Migration assay ............................................................................................................................. 97
FUTURE DIRECTIONS ................................................................................................ 97
What is the mechanism by which PAX3-FOXO1A alters expression of miRNAs?98
Is PAX3-FOXO1a regulation of miRNAs clinically significant? ................................ 99
CONCLUSIONS ........................................................................................................ 100
ACKNOWLEDGEMENTS .......................................................................................... 102
FIGURES ................................................................................................................. 103

REFERENCES ...................................................................................................................... 104

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LIST OF FIGURES
FIGURE 1. EMBRYONIC MYOGENESIS ............................................................................................. 27
FIGURE 2. MOLECULAR REGULATION OF EMBRYONIC MYOGENESIS. ............................................ 28
FIGURE 3. DIFFERENTIATION AND MYOMIR ANALYSIS IN CULTURED PRIMARY SKMCS .............. 45
FIGURE 4.DIFFERENTIAL EXPRESSION OF MIRS IN DIFFERENTIATED AND UNDIFFERENTIATED
SKMCS .................................................................................................................................. 46
FIGURE 5. COMPARISON OF MYOMIR EXPRESSION IN NORMAL SKMCS AND RMS CELL LINES ... 47
FIGURE 6. HIERARCHICAL CLUSTERING OF SKMCS AND RMS CELLS BASED ON MIRNA
EXPRESSION PROFILES ........................................................................................................... 48
FIGURE 7. SCHEMATIC OF COMPARISON BETWEEN DIFFERENTIATED AND UNDIFFERENTIATED
SKMCS TO ERMS AND ARMS CELLS .................................................................................. 49
FIGURE 8. MIRNAS DIFFERENTIALLY EXPRESSED BETWEEN NORMAL CELLS AND RMS CELL
LINES. .................................................................................................................................... 50
FIGURE 9. MYOMIR EXPRESSION IN ERMS CELL LINE RD VS. ARMS CELL LINE RH30 ............... 51
FIGURE 10. SCHEMATIC OF RD VS. RH30 MIRNA EXPRESSION ................................................... 52
FIGURE 11. DIFFERENTIALLY REGULATED MIRNAS IN ERMS VS. ARMS ................................... 53
FIGURE 12. CREATION AND ANALYSIS OF ENGINEERED CELL LINES. ............................................ 85
FIGURE 13. RNA-SEQ DATA FOR CANDIDATE MIRNAS ................................................................. 86
FIGURE 14. QPCR ANALYSIS ON PAX3-FOXO1A INDUCED MIRNAS .......................................... 87
FIGURE 15. DLK1-DIO3 REGION WITH ADJACENT MIR-495 AND MIR-543................................... 88
FIGURE 16. EFFECT OF PAX3-FOXO1A TRUNCATION ON MIRNA EXPRESSION ........................... 89
FIGURE 17. MIGRATION OF RMC CELLS WITH MIRNA INHIBITION. ............................................ 103

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