Università degli Studi di Milano Scuola di Dottorato in Medicina Molecolare Dipartimento di Scienze e Tecnologie Biomediche Curriculum di Genomica, Proteomica e Tecnologie Correlate Ciclo XXIV Settore Disciplinare: BIO-10 Anno Accademico 2010/2011 Dottorando: Dr.ssa Valentina TINAGLIA Matricola: R08079 INTEGRATED GENOMICS ANALYSIS OF GENE AND MICRORNA EXPRESSION PROFILES IN CLEAR CELL RENAL CARCINOMA CELL LINES Direttore della Scuola: Ch.mo Prof. Mario Clerici Tutore: Prof.ssa Cristina Battaglia Un grazie speciale a Mamma, Papà ed Enzo per la loro infinita pazienza e il loro amore. CONTENTS SOMMARIO .................................................................................................................................... V ABSTRACT .................................................................................................................................. VII 1 INTRODUCTION ...................................................................................................................... 1 1.1 Renal Cell Carcinoma ...................................................................................................... 1 1.1.1 Epidemiology ........................................................................................................... 1 1.1.2 Clinical features ....................................................................................................... 1 1.1.3 Clinical cytogenetic and molecular characteristics of renal tumors .................... 3 1.1.3.1 Familial renal cell carcinoma .............................................................................................. 5 1.1.3.1.1 VHL gene .......................................................................................................................... 6 1.1.3.1.2 Other genes implicated in ccRCC ..................................................................................... 9 1.1.4 Clinical utility of molecular profiling in cancer .................................................... 11 1.1.5 Gene expression studies ...................................................................................... 12 1.1.6 Signaling pathways in renal cell carcinoma and therapy ................................... 13 1.2 MICRORNA ..................................................................................................................... 17 1.2.1 microRNAs and mechanism of action .................................................................. 17 1.2.2 Genomic localization ............................................................................................. 18 1.2.3 miRNA biogenesis ................................................................................................. 19 1.2.4 Target prediction and new miRNAs discovered .................................................. 20 1.2.5 miRNA and cancer ................................................................................................. 22 1.2.6 Methods to detect miRNA expression .................................................................. 25 1.2.7 miRNA and RCC ..................................................................................................... 25 1.3 Integrative analysis of miRNA and mRNA expression profiles .................................. 29 1.3.1 Integrative genomics approach ............................................................................ 29 1.3.2 miRNA-mRNA integrated studies ......................................................................... 29 2 AIM OF THE STUDY .............................................................................................................. 33 3 PART I: HIGH-THROUGHPUT GENE EXPRESSION PROFILING ....................................... 35 3.1 Material and Methods..................................................................................................... 35 I 3.1.1 Cell Lines ................................................................................................................ 35 3.1.2 Total RNA and DNA extraction ............................................................................. 35 3.1.3 Assessment of VHL and HIF status ...................................................................... 35 3.1.4 High-throughput gene expression analysis ......................................................... 36 3.1.4.1 Target sample preparation for microarray gene expression analysis ......................... 36 3.1.4.2 Differential gene expression analysis ............................................................................. 38 3.1.4.3 Bioinformatics and functional enrichment analysis ...................................................... 38 3.2 Results ............................................................................................................................ 39 3.2.1 Assessment of VHL and HIF status ...................................................................... 39 3.2.2 High-throughput gene expression analysis ......................................................... 39 3.2.2.1 Differential gene profiling of Caki-1 vs HK-2 .................................................................. 39 3.2.2.2 Differential gene profiling of Caki-2 vs HK-2 .................................................................. 40 3.2.2.3 Differential gene profiling of A498 vs HK-2 ..................................................................... 41 3.3 Discussion ...................................................................................................................... 45 4 PART II: HIGH-THROUGHPUT MIRNA EXPRESSION PROFILING ..................................... 51 4.1 Material and Methods..................................................................................................... 51 4.1.1 High-throughput miRNA expression analysis ..................................................... 51 4.1.1.1 Target sample preparation for microarray miRNA expression analysis ..................... 51 4.1.1.2 Differential miRNA expression analysis .......................................................................... 52 4.1.1.3 Bioinformatics and functional enrichment analysis ...................................................... 53 4.1.1.4 Data validation by qPCR ................................................................................................... 53 4.2 Results ............................................................................................................................ 54 4.2.1 High-throughput miRNA expression analysis ..................................................... 54 4.2.1.1 Comparison between RCC cell lines and HK-2 .............................................................. 54 4.2.1.2 Functional enrichment analysis and validation by qPCR ............................................. 60 4.3 Discussion ...................................................................................................................... 62 5 PART III: INTEGRATED ANALYSIS OF MICRORNA AND GENE EXPRESSION DATA ..... 69 5.1 Material and Methods..................................................................................................... 69 5.1.1 miRNA-gene integrated analysis .......................................................................... 69 II 5.1.2 Selection of relevant miRNA-gene anti-correlated pairs ..................................... 69 5.1.3 qPCR validation of selected target genes ............................................................ 69 5.2 Results ............................................................................................................................ 71 5.2.1 miRNA-gene integrated analysis .......................................................................... 71 5.2.2 qPCR validation of selected target genes ............................................................ 73 5.3 Discussion ...................................................................................................................... 75 6 CONCLUSIONS AND FUTURE PROSPECTS ...................................................................... 81 7 BIBLIOGRAPHY .................................................................................................................... 83 8 GLOSSARY OF ABBREVIATIONS ....................................................................................... 97 9 APPENDIX ............................................................................................................................. 99 10 ACKNOWLEDGEMENTS ................................................................................................. 113 11 SCIENTIFIC PRODUCTS ................................................................................................. 115 III IV SOMMARIO Il carcinoma renale (RCC) è la neoplasia più frequente tra quelle che colpiscono il rene in età adulta e rappresenta il 2-3% di tutti i carcinomi umani. Il carcinoma renale a cellule chiare (ccRCC) è l‟istotipo più frequente (75-80%), invasivo e metastatico tra i sottotipi di RCC. Il gene von Hippel-Lindau (VHL), che è il principale oncosoppressore coinvolto negli stadi iniziali della tumorigenesi dell‟RCC, è soggetto ad inattivazione completa, per effetto combinato di mutazioni, delezioni e metilazione del promotore, nella maggior parte dei ccRCC sporadici e in tutte le forme ereditarie. Definire la prognosi dei casi di carcinoma renale è importante per operare scelte cliniche riguardo il trattamento dei pazienti, ma a volte la diagnosi differenziale risulta piuttosto difficile perché gli