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Celina Maria Dos Reis Parreira 2013

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Celina Maria Dos Reis Parreira 2013 DEPARTAMENTO DE CIÊNCIAS DA VIDA FACULDADE DE CIÊNCIAS E TECNOLOGIA UNIVERSIDADE DE COIMBRA The role of miR-21 in the bone marrow microenvironment Dissertação apresentada à Universidade de Coimbra para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biologia Celular e Molecular, realizada sob a orientação científica do Professor Doutor Sérgio Dias (Universidade de Lisboa) e do Professor Doutor Carlos Duarte (Universidade de Coimbra). Celina Maria dos Reis Parreira 2013 1 2 Acknowledgements First of all, I would like to express my gratitude to Doctor Sérgio Dias for accepting to be my supervisor and for giving me the opportunity to carry out my master dissertation with the excellent group that he coordinates. I am grateful for all the knowledge transmitted and for the confidence placed in me. I would also like to thank my co-supervisor, Doctor Carlos Duarte, for the understanding and interest and also the commitment dedicated to the master in Cellular and Molecular Biology. I would like to thank the coordinator of the master in Cellular and Molecular Biology, Doctor Emília Duarte, for her commitment to the course and also for her willingness and useful discussions. I thank all my colleagues from the laboratory, for all the transmitted knowledge, support, help, advices and good moments shared in the lab. I want to thank specially Joana Afonso who was always available to help me, for the encouragement, friendship, confidence and the knowledge shared with me. I would also like to thank my master colleagues, especially João, Andreia and Raquel, for the share of difficulties throughout this year. I would like to thank my friends for the support, advices, smiles, everything. To those who, despite being far away, have always been close. I want also to thank people of Republica77, especially Patrícia, Sandra e Fernas, for all the good moments, friendship, complicity and support. À minha família, um agradecimento muito especial por tudo. Aos meus tios e primos e aos meus avós pela compreensão, confiança e apoio que sempre me deram. Não posso também deixar de agradecer ao meu irmão, Nuno, pelo companheirismo, apoio, preocupação e pelas brincadeiras. Aos meus pais pelas oportunidades que me deram, por acreditarem em mim, pela força e apoio incondicionais, por serem o meu “porto” seguro. Obrigada por tudo, principalmente pelos valores e sábios conselhos. 3 4 Index Index ............................................................................................................................. 5 Illustrations Index ......................................................................................................... 7 Figures .................................................................................................................................. 7 Tables ................................................................................................................................... 9 Abbreviations .............................................................................................................. 10 Abstract ...................................................................................................................... 12 Resumo ....................................................................................................................... 13 Introduction ................................................................................................................ 15 Blood vessels and endothelial cell properties ...................................................................... 15 1. Blood vessels ........................................................................................................... 15 2. Endothelial Cells ...................................................................................................... 16 2.1. Endothelial Cell Polarity ................................................................................... 17 2.2. Endothelial Cell Adhesion ................................................................................. 17 2.2.1. Cell-to-cell Junctions ................................................................................ 19 2.2.2. Cell-to-matrix Junctions ............................................................................ 20 2.3. Endothelial cells proliferation and apoptosis .................................................... 21 3. Angiogenesis ........................................................................................................... 21 Bone marrow vessels: “instructors” of hematopoietic function ........................................... 23 MicroRNAs .......................................................................................................................... 25 1. miRNA biogenesis and mechanism of action ............................................................ 25 2. miRNA nomenclature .............................................................................................. 26 3. Prediction of miRNA putative targets ....................................................................... 27 MicroRNA-21 ...................................................................................................................... 29 Aims ............................................................................................................................ 32 Methods ..................................................................................................................... 33 Cell Culture ......................................................................................................................... 33 HUVECs Transfection........................................................................................................... 33 RNA Extraction .................................................................................................................... 34 cDNA Synthesis ................................................................................................................... 35 1. cDNA Synthesis for analysis of miRNA levels ............................................................ 35 2. cDNA Synthesis for analysis of expression of coding genes ....................................... 36 5 Real-time Quantitative PCR (RT-qPCR) ................................................................................. 36 1. Quantification of miRNA levels ................................................................................ 37 2. Quantification of coding genes ................................................................................ 37 Matrigel Assay .................................................................................................................... 39 “Wound healing/Scratch” Assay .......................................................................................... 39 Mice Genotyping ................................................................................................................. 39 Statistical Analysis ............................................................................................................... 41 Results and Discussion ................................................................................................ 42 miR-21 expression on bone marrow cells ............................................................................ 42 hsa-miR-21-5p predicted targets ......................................................................................... 43 1. Angiocrine factors.................................................................................................... 43 2. Angiogenic factors ................................................................................................... 45 2.1. ANXA1 ............................................................................................................. 45 2.2. IL-15................................................................................................................. 46 2.3. PTEN ................................................................................................................ 47 2.4. RECK ................................................................................................................ 47 2.5. RhoB ................................................................................................................ 47 2.6. Spry1 and Spry2 ............................................................................................... 47 2.7. TSP-1 ............................................................................................................... 48 miR-21 modulation of its predicted targets ......................................................................... 51 RT-qPCR different approaches ......................................................................................... 51 Validation of mir-21 predicted targets ............................................................................. 53 Phenotypic changes modulated by miR-21 levels: Tube formation assay ............................. 57 Phenotypic changes modulated by miR-21 levels: Cell migration assay................................ 59 Endothelial-specific miR-21 KO mice generation.................................................................. 61 Conclusions ................................................................................................................. 65 References .................................................................................................................
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