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Micrornas and Their Processing Factors in Caenorhabditis Elegans and Human Cancers

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Micrornas and Their Processing Factors in Caenorhabditis Elegans and Human Cancers From DEPARTMENT OF ONCOLOGY-PATHOLOGY Karolinska Institutet, Stockholm, Sweden MICRORNAS AND THEIR PROCESSING FACTORS IN CAENORHABDITIS ELEGANS AND HUMAN CANCERS Roger Kae-Jia Chang Stockholm 2018 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by AJ E-print AB, 2018 © Roger Kae-Jia Chang, 2018 ISBN 978-91-7676-929-4 MicroRNAs and their processing factors in Caenorhabditis elegans and human cancers THESIS FOR DOCTORAL DEGREE (Ph.D.) By Roger Kae-Jia Chang Principal Supervisor: Opponent: Weng-Onn Lui, Associate Professor Carlos Rovira, Associate Professor Karolinska Institutet Lund University Department of Oncology-Pathology Department of Clinical Sciences Co-supervisor(s): Examination Board: Klas Wiman, Professor Neus Visa, Professor Karolinska Institutet Stockholm University Department of Oncology-Pathology Department of Molecular Biosciences Andrea Hinas, Assistant Professor Uppsala University Department of Cell and Molecular Biology Angelo De Milito, Associate Professor Karolinska Institutet Department of Oncology-Pathology To my friends and family, this would not be possible without you! It is difficult to sum up the contributions all of you made into realizing this thesis. I am grateful for your encouragement and support for allowing me to follow science. To borrow the words of Sir. Isaac Newton: “If I have seen further, it is by standing upon the shoulders of giants”. Thanks for letting my see further than I could imagine. Thanks for letting me stand on your shoulders. ABSTRACT MicroRNAs (miRNAs) are small noncoding RNAs approximately 20-22 nucleotides (nt) long. These small RNAs (sRNAs) was initially discovered in Caenorhabditis elegans (C. elegans), but is conserved in over 50 animal species. According to miRbase, a database of miRNA sequences from various species, humans have over 2500 different miRNAs and estimated to regulate over 60% of the protein coding genes in humans. Due to their role in gene regulation, they are important for controlling key processes in the cell, and changes in miRNA expression in cell can have catastrophic consequences. In this thesis we explore the role of miRNA and their biogenesis factor. We begin by studying the first miRNA to be identified in humans, let.7. We use the C. elegans model system with different genetic strains to describe an unknown role for this miRNA. The unravelling of this new role highlights the importance of miRNA as important regulator in cells not only for protein coding transcripts but also transcripts that do not code for proteins. The findings of this study opens up new and exciting research possibilities for miRNAs. Because of miRNAs importance in controlling key cellular processes in cells. Changes in their expression patterns can lead to severe effect. In study II we explore one such event, development of human tumors. We characterize the miRNA profile of different types of ovarian tumors to identify biomarkers that can be used in clinics and to understand the pathology of the tumors to find an effective cure. Using only a few miRNAs we can characterize different types of ovarian tumors based on their miRNA profile. We also identify a miRNA target that could potentially be explored for therapy. The miRNA biogenesis factors are important to ensure proper production of miRNAs. Their role in the miRNA biogenesis is well characterized, but very little is known if they possess other biological function besides miRNA production. In study III we describe a new role for TARBP2, which is responsible for miRNA maturation. Hundreds of thousands RNAs are transcribed from a single cell, and each RNA molecule have different sequences or structures that are important for their function/regulation. We used computational biology to predict all the potential TARBP2-interaction sites on messenger RNAs, and found several in the 5’ untranslated region (5’UTR). The 5’UTR are just upstream of the initiation site of protein synthesis, and usually contain important regulatory sequences. We explore how the TARBP2 interaction with the 5’UTRs can affect gene expression. In particularly we found a gene that is important for regulation of a process in the cell called autophagy. In this thesis we investigate the role of miRNAs and their processing factors in C. elegans and humans. We describe a new role for miRNA as regulator of noncoding transcript, and we explore the possibility of using them as biomarkers in ovarian tumors. Finally, we describe a new role for the miRNA biogenesis factor TARBP2 as a regulator of autophagy. LIST OF SCIENTIFIC PAPERS The thesis is based on the following papers, which will be referred throughout the text by their Roman numerals. I. Zisoulis DG, Kai ZS, Chang RK, Pasquinelli AE. Autoregulation of microRNA biogenesis by let-7 and Argonaute. Nature, 486:541-4 (2012) II. Chang RK, Li X, Mu N, Hrydziusko O, Garcia-Majano B, Larsson C, Lui WO. MicroRNA expression profiles in non-epithelial ovarian tumors. Int J Oncol., 52:55-66 (2018) III. Chang RK, Gao J, Li X, Garcia-Majano B, Wiman K, Lui WO. TARBP2 regulates autophagy through post-transcriptional regulation of ATG2A expression. Manuscript CONTENTS 1 Caenorhabditis elegans as a biological model system .................................................. 1 1.1 Life cycle of C. elegans ......................................................................................... 2 2 microRNA ....................................................................................................................... 4 2.1.1 lin-4, the first miRNA discovered in C. elegans ...................................... 4 2.1.2 let-7, the first miRNA discovered in humans ........................................... 4 2.2 miRNA biogenesis pathway .................................................................................. 5 2.2.1 The non-canonical miRNA biogenesis pathway ...................................... 5 6 2.3 Mechanisms of miRNA regulation ....................................................................... 6 2.3.1 mRNA degradation ................................................................................... 6 2.3.2 Translational repression ............................................................................ 7 3 TARBP2, a RNA binding protein involved in miRNA biogenesis ............................... 8 4 Autophagy ..................................................................................................................... 10 4.1 mTORC1 a regulator of autophagy .................................................................... 10 4.2 Autophagosome biogenesis ................................................................................. 10 4.2.1 Initiation................................................................................................... 10 4.2.2 Nucleation................................................................................................ 11 4.2.3 Expansion ................................................................................................ 11 5 Aims of the study .......................................................................................................... 13 5.1 Study I: ................................................................................................................. 13 5.2 Study II: ............................................................................................................... 13 5.3 Study III: .............................................................................................................. 13 6 MATERIALS AND METHODS ................................................................................. 15 6.1 Nematode culture and strains .............................................................................. 15 6.1.1 MosSCI .................................................................................................... 15 6.1.2 Crossing ................................................................................................... 15 6.2 RNAi treatment in C. elegans ............................................................................. 17 6.3 RNA detection methods ...................................................................................... 17 6.3.1 Northern Blotting .................................................................................... 17 6.3.2 RT-PCR ................................................................................................... 18 6.3.3 Sequence-based approach ....................................................................... 19 6.4 Bioinformatical analysis ...................................................................................... 21 6.4.1 Burrows-Wheeler alignment ................................................................... 23 6.4.2 Tophat2, discovering splice junctions .................................................... 23 6.5 Transfection of DNA or RNA into mammalian cells ........................................ 23 6.5.1 Lipid-based transfection .......................................................................... 23 6.5.2 Polyamine-based transfection ................................................................. 23 6.6 Protein-RNA interaction ..................................................................................... 23 6.7 Monitoring autophagy in mammalian cells ........................................................ 25 6.7.1 Monitoring LC3 turnover by immunoblotting ......................................
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