Posttranscriptional regulation of the components of the human leukocyte antigen class I antigen processing and presentation machinery as an immune escape mechanism in melanoma Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg, vorgelegt von Frau Maria-Filothei Lazaridou geb. am 29.04.1991 in Athen (Griechenland) Gutachter: 1. Prof. Dr. Stefan Hüttelmaier, Institut für Molekulare Medizin, Martin-Luther- Universität Halle-Wittenberg 2. Prof. Dr. Barbara Seliger, Institut für Medizinische Immunologie, Martin-Luther- Universität Halle-Wittenberg 3. Prof. Dr. Ourania E. Tsitsilonis, Department of Animal and Human Physiology, Faculty of Biology, National and Kapodistrian University of Athens Tag der öffentlichen Verteidigung: 07.04.2021 Contents Table of contents 1. Introduction ............................................................................................................................ 1 1.1 Melanoma ........................................................................................................................ 1 1.2 The immune system ........................................................................................................ 5 1.3 Characteristics of MHC-I molecules and the antigen processing and presentation machinery (APM) ................................................................................................................... 7 1.4 Defects of MHC-I expression as an immune escape mechanism................................. 14 1.5 Posttranscriptional regulation of gene expression ........................................................ 15 1.5.1 MicroRNAs .............................................................................................................. 16 1.5.2 RNA binding proteins .............................................................................................. 20 2. Aims ..................................................................................................................................... 23 3. Materials .............................................................................................................................. 24 3.1 Chemicals ...................................................................................................................... 24 3.2 Buffers ........................................................................................................................... 26 3.3 Oligonucleotides ............................................................................................................ 27 3.4 Antibodies ...................................................................................................................... 31 3.5 Enzymes ........................................................................................................................ 31 3.6 Kits ................................................................................................................................. 32 3.7 Consumables ................................................................................................................. 33 3.8 Equipment ...................................................................................................................... 34 3.9 Software ......................................................................................................................... 34 4. Methods ............................................................................................................................... 36 4.1 Cell culture conditions and cell lines ............................................................................. 36 4.2 Transient transfection .................................................................................................... 37 4.3 Isolation of DNA, RNA and miRs ................................................................................... 37 4.4 cDNA synthesis and quantitative qPCR ........................................................................ 37 4.5 Protein extraction and measurement of protein concentration ...................................... 38 4.6 Western blot analysis .................................................................................................... 38 4.7 Flow cytometric analysis ................................................................................................ 39 4.8 CD107 degranulation assays ........................................................................................ 39 4.9 Dual luciferase reporter assay ....................................................................................... 39 4.10 MiRNA trapping by RNA in vitro affinity purification (miTRAP) assay ......................... 40 4.11 Small RNA sequencing analysis ................................................................................. 42 4.12 RNA affinity purification for the detection of RNA-binding proteins ............................. 43 4.13 Colloidal Coomassie staining ...................................................................................... 45 4.14 Protein band picking and sample preparation for mass spectrometry analysis .......... 45 4.15 Mass spectrometry analysis ........................................................................................ 45 4.16 Immunohistochemical staining of the paraffin-embedded tissue sections of melanoma patients ................................................................................................................................ 45 I Contents 4.17 Functional and pathway enrichment analyses ............................................................ 46 4.18 Survival analysis of clinical data of tumour patients .................................................... 46 4.19 Statistical analysis ....................................................................................................... 47 5. Results ................................................................................................................................. 48 5.1 Heterogeneous expression of HLA-I APM components in melanoma cell lines ........... 48 5.2 Clinical relevance of APM components and HLA-I molecules with survival of metastatic melanoma patients .............................................................................................................. 52 5.3 Identification of immune modulatory miRs by the miTRAP assay in melanoma cell lines ............................................................................................................................................. 52 5.4 Expression of the strongly enriched candidate miRs in melanoma cell lines ................ 58 5.5 Proof of direct interaction of the newly identified regulating miRs with the TAP1 3’-UTR by the dual luciferase reporter assay................................................................................... 62 5.5.1 Prediction of the minimum free energy for the candidate miRs and TAP1 3’-UTR by the in silico RNAhybrid tool .............................................................................................. 62 5.5.2 Functional validation of candidate miRs via dual luciferase reporter assays ......... 62 5.6 Effects of the overexpression of the candidate miRs on HLA-I APM component expression ........................................................................................................................... 64 5.7 Correlation between the miR-mediated downregulation of TAP1 protein expression and its effect on the recognition by NK or T-cells ....................................................................... 68 5.7.1 Effect of the miR-200a-5p-mediated downregulation of TAP1 protein expression on the recognition by NK cells .............................................................................................. 68 5.7.2 Correlation of the miR26b-5p-mediated downregulation of TAP1 with decreased T- cell recognition ................................................................................................................. 69 5.8 Effects of the miR-26b-5p- or miR-21-3p-mediated inhibition on theTAP1 expression and HLA-I surface expression pattern ................................................................................. 70 5.9 Correlations between miRs expression pattern and corresponding TAP1 expression levels as well as immune cell infiltration in primary melanoma tissues ............................... 71 5.10 Associations of the expression pattern of the candidate miRs with clinical parameters and in particular with the survival of melanoma patients ..................................................... 74 5.11 Identification of immune modulatory RBPs by RNA-AP assays in melanoma cell lines ............................................................................................................................................. 75 5.12 Expression of the enriched candidate RBPs in melanoma cell lines ...................... 78 5.13 Correlation between the mRNA expression levels of enriched candidate RBPs and TPN as well as the survival rates of melanoma patients ..................................................... 79 5.14 Effect of the deregulation of the RBPs IGF2BP1 and IGF2BP3 on the expression pattern of HLA-I APM components ...................................................................................... 80 5.15 Identification of deregulated proteins upon knock down or overexpression