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Micro-Rnas in Ovarian Cancer As Tools for Diagnosis and Therapy Blanca ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Micro-RNAs in ovarian cancer as tools for diagnosis and therapy Blanca Majem Cavaller Blanca Majem Cavaller June 27th, 2017 Micro-RNAs in ovarian cancer as tools for diagnosis and therapy PhD thesis presented by Blanca Majem Cavaller To obtain the degree of PhD for the Universitat Autònoma de Barcelona (UAB) PhD thesis done at the Cell Cycle and Cancer laboratory, within the Biomedical Research Group in Gynecology, at Vall d'Hebron Research Institute (VHIR) and University Hospital (HUVH), under the supervision of Dr. Anna Santamaria, Dr. Marina Rigau and Dr. Marta Llauradó Thesis affiliated to the Department of Cell Biology, Physiology and Immunology from the School of Medicine at UAB, in the PhD program of Cell Biology, under the tutoring of Dr. Rosa Miró Universitat Autònoma de Barcelona, June 27th 2017 Dr. Anna Santamaria Dr. Marina Rigau Dr. Marta Llauradó (Director) (Director) (Director) Dr. Rosa Miró Blanca Majem Cavaller (Tutor) (Student) “The triumph is not always to conquer but never to be discouraged” (Anonymous) "Courage is not the absence of fear, but the triumph over it” (Nelson Mandela) Index INDEX ABBREVIATIONS ....................................................................................................... 13 1. INTRODUCTION ..................................................................................................... 17 1.1. OVARIAN CANCER GENERALITIES .......................................................................... 17 1.1.1. EPIDEMIOLOGY .................................................................................................... 18 1.1.2. STAGING AND SURVIVAL .................................................................................... 18 1.1.3. HISTOLOGY ........................................................................................................... 21 1.1.4. RISK FACTORS ..................................................................................................... 25 1.1.5. ETIOLOGY ............................................................................................................. 27 1.2. OVARIAN CANCER DIAGNOSIS ................................................................................ 28 1.2.1. BACKGROUND ...................................................................................................... 28 1.2.2. CURRENT DIAGNOSTIC TOOLS ......................................................................... 30 1.2.3. SCREENING TOOLS AND DIAGNOSTIC BIOMARKERS .................................... 33 1.2.4. SALIVA AS SOURCE OF BIOMARKERS .............................................................. 38 1.3. OVARIAN CANCER THERAPY ................................................................................... 43 1.3.1. BACKGROUND ...................................................................................................... 43 1.3.2. CURRENT THERAPEUTIC TOOLS ...................................................................... 43 1.3.3. TARGETED THERAPIES ....................................................................................... 49 1.4. MIRNAS: NOVEL STRATEGIES FOR DIAGNOSIS AND THERAPY ......................... 53 1.4.1. MiRNA MOLECULES ............................................................................................. 53 1.4.2. MiRNAs IN OVARIAN CANCER ............................................................................ 60 1.4.2.1. MiRNAs as a diagnostic tool .......................................................................................... 63 1.4.2.2. MiRNAs as a therapeutic tool ........................................................................................ 66 2. HYPOTHESIS AND OBJECTIVES ......................................................................... 71 3. MATERIALS AND METHODS ................................................................................ 73 3.1. MATERIALS .................................................................................................................. 73 3.1.1. HUMAN OVARIAN CANCER SAMPLES ............................................................... 73 3.1.2. CELL CULTURES .................................................................................................. 77 3.1.3. ANTIBODIES .......................................................................................................... 79 3.1.4. PRIMERS AND PROBES ....................................................................................... 79 3.1.5. OLIGONUCLEOTIDES ........................................................................................... 80 3.2. METHODS ..................................................................................................................... 81 3.2.1. HIGH-THOUGHPUT DATA .................................................................................... 81 3.2.1.1. Small RNA sequencing analysis of saliva ...................................................................... 81 9 3.2.1.2. Human miRNA microarray analysis ............................................................................... 82 3.2.1.3. Human mRNA microarray analysis ................................................................................ 83 3.2.1.4. DNA methylation microarray analysis ............................................................................ 84 3.2.2. IN VITRO EXPERIMENTS ..................................................................................... 84 3.2.2.1. Transient transfections .................................................................................................. 84 3.2.2.2. Constructs generation .................................................................................................... 85 3.2.2.3. Lentivirus production and transduction .......................................................................... 86 3.2.2.4. Protein extraction and Western Blot .............................................................................. 86 3.2.2.5. Immunohistochemistry ................................................................................................... 87 3.2.2.6. RNA extractions and RTqPCR ...................................................................................... 87 3.2.2.7. Proliferation assay ......................................................................................................... 88 3.2.2.8. Cell death assay ............................................................................................................ 89 3.2.2.9. Cell cycle assay ............................................................................................................. 89 3.2.2.10. DNA demethylating assay ........................................................................................... 90 3.2.2.11. 3’UTR luciferase reporter assay .................................................................................. 90 3.2.2.12. Spheroid formation assays .......................................................................................... 90 3.2.3. IN VIVO EXPERIMENTS ........................................................................................ 92 3.2.4. STATISTICAL ANALYSIS ...................................................................................... 93 3.2.4.1. Salivary biomarkers data ............................................................................................... 93 3.2.4.2. MiRNA array data from FFTE tumors ............................................................................ 94 3.2.4.3. Microarray analysis of CDCP1 and PLAGL2 silencing .................................................. 94 3.2.4.4. Human samples, in vitro and in vivo assays .................................................................. 95 3.2.5. BIOINFORMATIC TOOLS ...................................................................................... 95 4. RESULTS ................................................................................................................ 97 4.1. MIRNAS IN SALIVA AS A DIAGNOSTIC TOOL ......................................................... 97 4.1.1. Salivary RNA from HGSC and benign patients are suitable for RNA sequencing . 97 4.1.2. Small RNA sequencing revealed increased levels of new miRNA in saliva from HGSC patients ................................................................................................................ 104 4.2. MIRNAS AS A TOOL FOR OC TREATMENT ............................................................ 109 4.2.1. Identification of 4 deregulated miRNAs in advanced stage OC patients .............. 109 4.2.2. Evaluation of the oncogenic/tumor suppressor role of the validates miRNAs ...... 111 4.2.3. MiR-654 levels were decreased in OC tumor samples ........................................ 111 4.2.4. MiR-654 overexpression reduced cell proliferation and induced apoptotic cell death .......................................................................................................................................
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