Aus dem Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Christian Weber WISP1 Regulation by MicroRNAs in Pulmonary Fibrosis Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Barbara Berschneider aus Regensburg 2014 Meiner Familie Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Betreuer: PD Dr. rer. nat. Peter Neth Zweitgutachter: Prof. Dr. rer.nat. Wolfgang Zimmermann Dekan: Prof. Dr.med. Dr.h.c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung: 6. November 2014 TABLE OF CONTENTS Table of Contents ........................................................................................................... I Abbreviations .............................................................................................................. VI 1 Zusammenfassung .................................................................................................. 1 2 Summary .................................................................................................................... 2 3 Introduction ............................................................................................................. 3 3.1 Idiopathic pulmonary fibrosis ............................................................................................................... 3 3.1.1 Clinical features of IPF .................................................................................................................... 3 3.1.2 Pathological features of IPF .......................................................................................................... 4 3.1.3 Pathomechanisms of IPF ............................................................................................................... 5 3.1.3.1 Genetic predispositions of pulmonary fibrosis ........................................................... 6 3.1.3.2 Key cell types in pulmonary fibrosis ................................................................................ 6 3.1.3.3 Growth factor signalling in pulmonary fibrosis .......................................................... 7 3.1.4 Animal models of lung fibrosis ................................................................................................... 9 3.1.4.1 Bleomycin model of pulmonary fibrosis ........................................................................ 9 3.1.4.2 Overexpression of TGF-β1 .................................................................................................... 9 3.2 WISP1 ............................................................................................................................................................ 10 3.2.1 WISP1 gene and protein structure ......................................................................................... 10 3.2.2 WISP1 expression .......................................................................................................................... 11 3.2.3 WISP1 function ............................................................................................................................... 12 3.2.4 WISP1 regulation ........................................................................................................................... 14 3.3 MicroRNAs .................................................................................................................................................. 15 3.3.1 MiRNA biogenesis .......................................................................................................................... 15 3.3.2 MiRNA target recognition and function ............................................................................... 17 3.3.3 MiRNAs in pulmonary fibrosis ................................................................................................. 18 3.4 Aims of this study ..................................................................................................................................... 19 I 4 Materials and Methods ....................................................................................... 20 4.1 Materials ...................................................................................................................................................... 20 4.1.1 Laboratory equipment and software .................................................................................... 20 4.1.2 Chemicals and consumables ..................................................................................................... 22 4.1.3 Buffers and solutions ................................................................................................................... 24 4.1.4 Standards and kits ......................................................................................................................... 25 4.1.5 Enzymes ............................................................................................................................................. 26 4.1.6 Plasmids ............................................................................................................................................ 27 4.1.6.1 Molecular cloning .................................................................................................................. 28 4.1.6.2 SiRNA .......................................................................................................................................... 29 4.1.6.3 Sequencing primers .............................................................................................................. 30 4.1.6.4 MiRNA – mimics and inhibitors ...................................................................................... 30 4.1.6.5 MiScript primer assays ....................................................................................................... 31 4.1.6.6 TaqMan assays ........................................................................................................................ 31 4.1.6.7 Quantitative PCR .................................................................................................................... 31 4.1.7 Antibodies ......................................................................................................................................... 33 4.1.8 Bacteria .............................................................................................................................................. 34 4.1.9 Cell lines and primary cells ....................................................................................................... 35 4.1.10 Animals ............................................................................................................................................... 36 4.1.11 Human Tissue .................................................................................................................................. 36 4.2 Methods ........................................................................................................................................................ 37 4.2.1 Animal models of pulmonary fibrosis .................................................................................. 37 4.2.1.1 Bleomycin model of pulmonary fibrosis ..................................................................... 37 4.2.1.2 Adenoviral TGF-β1-induced fibrosis ............................................................................. 37 4.2.2 Cell biology ....................................................................................................................................... 38 4.2.2.1 Primary cell isolations ......................................................................................................... 38 4.2.2.1.1 Isolation of primary alveolar type II cells .......................................................................... 38 4.2.2.1.2 Isolation of primary fibroblasts ............................................................................................. 38 4.2.2.2 Cryopreservation of mammalian cells ......................................................................... 39 4.2.2.3 Culturing and sub-culturing of mammalian cells .................................................... 39 4.2.2.4 Cell treatments ....................................................................................................................... 40 4.2.2.5 Lipotransfection of cells ..................................................................................................... 40 II 4.2.3 Microbiology .................................................................................................................................... 42 4.2.3.1 Transformation of chemically competent E. coli ..................................................... 42 4.2.3.2 Storage of E. coli ..................................................................................................................... 42 4.2.3.3 Cultivation of E. coli .............................................................................................................. 42 4.2.4 Molecular biology .......................................................................................................................... 42 4.2.4.1 Isolation of genomic DNA (gDNA) .................................................................................. 42 4.2.4.2