INAUGURAL – DISSERTATION zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprecht-Karls-Universität Heidelberg vorgelegt von Master of Research Kaiyong Li aus Stuttgart Tag der mündlichen Prüfung: . 2018 Identification of a functional mRNA-miRNA network in mTEC development Gutachter: Prof. Bruno Kyewski / Prof. Nina Papavasiliou Prof. Stefan Wiemann Hiermit erkläre ich, dass ich die vorgelegte Dissertation selbstständig verfasst und mich dabei keiner anderen, als der von mir ausdrücklich bezeichneten Quellen bedient habe. Stuttgart, den 18. Mai 2018 Kaiyong Li This thesis was completed in the Department of Developmental Immunology headed by Prof. Dr. Bruno Kyewski at the German Cancer Research Center Heidelberg Content Content Table of Contents Content ................................................................................................................................. 1 Zusammenfassung ............................................................................................................ 3 Summary .............................................................................................................................. 5 1 Introduction .................................................................................................................... 7 1.1 The evolution and development of the thymus ................................................. 7 1.1.2 The evolution of the adaptive immune system .............................................. 8 1.2.1 The maturation of thymocytes in the thymus ..................................................... 8 1.2.2 The selection of thymocytes and establishment of central tolerance ..... 9 1.2.3 The thymocyte selection model ............................................................................ 11 1.2.4 The discrepancy between cTECs and mTECs ................................................ 11 1.2.5 The maturation of mTECs ........................................................................................ 12 1.2.6 The crosstalk between developing thymocytes and thymus ................... 13 1.3 The Signalling pathways in TEC development ................................................... 14 1.4 microRNAs and their influence in post-translational regulation ................. 15 1.4.1 The involvement of microRNAs in signalling pathways ............................. 18 1.5 microRNAs in thymus development ....................................................................... 19 1.5.1 Functional miRNAs in mTECs ................................................................................ 20 1.1 Objective of this study ............................................................................................ 23 2 Materials and Methods .............................................................................................. 24 2.1 Materials .............................................................................................................................. 24 2.1.1 Chemicals ....................................................................................................................... 24 2.1.2 Dispensable materials and commercial kits .................................................... 26 2.1.3 Media, buffers and commercial solutions ......................................................... 28 2.1.4 Magnetic beads and Microbeads used for MACS purification ................. 30 2.1.5 Antibodies ...................................................................................................................... 31 2.1.6 MiRNA primers, siRNA, antagomiRs and mimics .......................................... 32 2.1.7 Oligonucleotides for vector cloning .................................................................... 35 2.1.8 PCR primers ................................................................................................................... 36 2.1.9 Equipment ...................................................................................................................... 37 2.1.10 Cell lines ....................................................................................................................... 38 2.1.11 Softwares and homepages ................................................................................... 39 2.1.12 Mouse strains ............................................................................................................. 39 2.2 Methods ....................................................................................................................... 41 2.2.1 Isolation of murine tail DNA .................................................................................... 41 2.2.2 Isolation of RNA from sorted cells or tissue .................................................... 41 2.2.3 Isolation of miRNA ...................................................................................................... 42 2.2.4 RNA precipitation ........................................................................................................ 42 2.2.5 MiRNA Reverse Transcription ................................................................................ 42 2.2.6 cDNA Synthesis ........................................................................................................... 42 2.2.7 Genotyping PCR ........................................................................................................... 42 2.2.8 Thymus dissection and medullary epithelial cell preparation ................. 43 1 Content 2.2.9 Thymus dissection and isolation of embryonic medullary thymic epithelial cells .......................................................................................................................... 44 2.2.10 Re-aggregate Thymus Organ Culture (RTOC) .............................................. 45 2.2.11 FACS staining of mTECs ........................................................................................ 46 2.2.12 Bone marrow transplantation .............................................................................. 46 2.2.13 Transient transfections with freshly isolated cells ..................................... 47 2.2.14 Vector generation, Transformation, Co-transfection ................................. 47 2.2.15 Luciferase assay ....................................................................................................... 48 3 Results ............................................................................................................................. 49 3.1 Four subpopulations within the immature mTEC pool ................................... 49 3.2 The four subpopulations in distinct mouse strains .......................................... 50 3.3 The mRNA expression profiling of the four subpopulations ........................ 53 3.4 The miRNA expression profiling of the four subpopulations ....................... 57 3.5 A developmental progression among the four subpopulations ................. 60 3.6 Finding the most prominent miRNAs in the development of immature mTECs ......................................................................................................................................... 62 3.7 Verification of the potential miRNAs in different mouse lines ..................... 63 3.8 A dynamic model to follow the mTEC development ........................................ 68 3.9 A dynamic model to reveal miRNA changes during mTEC development69 3.10 Discovery of dynamic miRNAs during mTEC development ....................... 73 3.11 Investigation of the functional role of our miRNA candidates ................... 75 3.12 Transfection methods on primary cells .............................................................. 76 3.13 Transfection efficacy of mimics and antagomiRs in mTECs ...................... 79 3.14 Further adjustments and optimization of cell numbers and controls .... 82 3.15 A change in the miRNA level has an impact in the four subpopulations ........................................................................................................................................................ 87 3.16 Search of mRNA targets ............................................................................................ 95 3.17 Quick screening of mRNA targets ......................................................................... 96 4 Discussion ................................................................................................................... 101 4.1 Identifying a lineage progression within the four subpopulations ......... 102 4.2 Clusters of miRNA sets in the four subpopulations ...................................... 104 4.3 Usage of different approaches to find potential miRNAs ........................... 106 4.4 Discovery of a new reliable transfection method ........................................... 108 4.5 MicroRNA level changes influence the development of the four subpopulations .................................................................................................................... 109 4.6 Search for relevant mRNA-miRNA pairs ............................................................ 110 4.7 MiRNAs with functional effects .............................................................................. 110 4.8 The new mRNA targets in immature mTECs and their potential involvement in the mTEC development ..................................................................... 111 4.9 Interesting mRNA target for further investigation