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Selenium-Gene Interactions Involving Micrornas

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Selenium-Gene Interactions Involving Micrornas Selenium-gene interactions involving microRNAs Anabel Maciel Domínguez Thesis submitted in partial fulfilment of the requirements of the regulations for the degree of Doctor of Philosophy Newcastle University Faculty of Medical Sciences Institute for Cell and Molecular Biosciences July / 2012 1 Declaration The material contained within this thesis has not been previously submitted for a degree at the University of Newcastle or any other university. The research here has been conducted by the author. All help by other has been acknowledged and all sources of information are indicated in the text. i Abstract Selenium (Se) is an essential nutrient for health. In mammals Se is incorporated into ~25 selenoproteins in the form of the amino-acid selenocysteine encoded by the UGA codon through a complex interacting with selenocysteine insertion sequence (SECIS) in the 3’Unstralated Region. The selenoproteins have functions in antioxidant defence and redox control, thyroid hormone metabolism and mitochondrial metabolism. Previous scientific work has found that Se also affects a group of downstream targets. The aim of my work is to investigate whether expression of selenoproteins or the downstream targets affected by Se is regulated through epigenetic mechanisms involving microRNAs, a small non-coding RNA species that regulates a gene or groups of genes by binding to the mRNA 3’UTR. Gut epithelial Caco-2 cells were grown in either Se deficient or Se-supplemented medium for 72h. RNA extracted and miRNA expression analysed using a custom-designed human genome V2 Agilent 8x15K array. In addition, global mRNA transcriptome expression was analysed using an Illumina HumanRef-8 v3 microarray. Se supply increased the expression of thirteen miRNAs and 53 mRNAs the observed differences were confirmed by real time PCR. miR- 185 was selected as a further target of investigation because of its high sensitivity to Se. Bioinformatic analysis of the Se susceptible miRNAs and Se sensitive genes was carried out using miRWalk, MicroCosmo, microRNA.org, miRBase and microRNA.org algorithms and Ingenuity Pathway Analysis. This identified miR-185 recognition elements in the Se- sensitive mRNAs for Glutathione peroxidise 2 (GPX2), Glutathione peroxidise 3 (GPX3), and Selenophosphate Synthetase 2 (SEPHS2). Expression of GPX2 and SEPHS2 was altered by miR-188 with a specific anti-miR and affected differently according to miRNA exposure. The data suggests that these mRNAs are targets of miR-185. In conclusion, the experiment indicates that miRNA expression as regulation of target genes is regulated by Se supply in Caco-2 cells. ii Acknowledgments Foremost all I would like to express my thanks and appreciation to Prof Hesketh for his patient guidance, encouragement, and support especially during though times of my project. I am thankful to have had a successful professor whose enthusiasm towards science has made my research experience stimulating and to have an excellent person as my supervisor. I would also like to show my gratitude to Prof Dianne Ford for her scientific contributions that pointed me in the right direction. I would also like to thank Dr Hannah Gautrey, Mr Chirs Blackwell, Dr Luisa Wakeling and Dr John Tyson for their patient and resourceful technical as well as personal contribution. This PhD thesis would not have been possible without the help of the Hesketh/Ford lab, where not only have I found good advice but also friendship. I would also like to acknowledge Dr Daniel Swan for conducting the statistical microarray data analysis, initial bioinformatics analysis and useful advice; to Dr Allison Howard and Dr David Young for letting me use their QRT-PCR facilities and for their guidance with the data analysis; to Dr Andy Verner for his constructive criticism and suggestions; to Dr Tim Cheek for looking after me on bureaucratic matters. I would also like to thank my examiners Dr Ruth Valentine and Prof Bernard Corfe for the time they invested reading my thesis and the insightful questions of my Viva voce. I am thankful to the National Council of Science and Technology who provided financial support for this research. Finally, I am truly in debt with my family, friends, and colleagues who contributed one way or another to this thesis, your love, support, and company have given me the strength to achieve this goal. Thank you all. iii "If we knew what it was we were doing, it wouldn't be called 'research,' would it?" Albert Einstein iv Table of Contents Declaration ................................................................................................... i Abstract ....................................................................................................... ii Acknowledgments ..................................................................................... iii Table of Contents ........................................................................................ v List of figures.............................................................................................. ix List of tables .............................................................................................. xii List of abbreviations ................................................................................. xiii Chapter 1 Introduction ............................................................................... 1 1.1 Introduction ....................................................................................... 1 1.2 Regulation of gene expression ......................................................... 2 1.2.1 Regulation of gene expression by nutrition ................................ 4 1.3 microRNAs (miRNAs) ....................................................................... 6 1.3.1 miRNA biogenesis in mammals ................................................. 9 1.3.2 Gene expression regulated by miRNAs ................................... 13 1.3.3 miRNA target recognition ......................................................... 15 1.3.4 Seed region ............................................................................. 16 1.3.5 Non-seeded sites ..................................................................... 18 1.3.6 miRNA conservation ................................................................ 18 1.3.7 mRNA target multiplicity ........................................................... 19 1.3.8 mRNA network regulation by a single miRNA .......................... 20 1.4 Bioinformatics approaches ............................................................. 21 1.4.1 Bioinformatic tools.................................................................... 22 1.4.2 Finding miRNAs through “hands in” laboratory techniques ...... 24 1.5 Selenium ........................................................................................ 28 1.5.1 Selenium and health ................................................................ 28 1.5.2 Food sources ........................................................................... 31 1.5.3 Organic or inorganic Se compounds? ...................................... 32 1.5.4 Selenoproteins ......................................................................... 34 1.5.5 Selenium uptake ...................................................................... 39 1.5.6 Selenoprotein biosynthesis ...................................................... 40 1.5.7 Downstream targets of Se ....................................................... 43 1.5.8 Selenium and colon ................................................................. 44 v 1.6 Aims and objectives ....................................................................... 47 Chapter 2 Materials and methods ............................................................ 50 2.1 Materials ......................................................................................... 50 2.1.1 Equipment ................................................................................ 50 2.1.2 Chemicals ................................................................................ 50 2.2 Methods .......................................................................................... 51 2.2.1 Cell culture ............................................................................... 51 2.2.2 Sodium selenite 7ng/mL; 40.5nM ............................................ 52 2.2.3 RNA isolation for total transcriptome analysis .......................... 52 2.2.4 Measurement of RNA Concentration ....................................... 54 2.2.5 RNA integrity assessment ........................................................ 54 2.2.6 miRNA target prediction for the protein biosynthetic pathway .. 55 2.2.7 Primer design ........................................................................... 62 2.2.8 Primer optimization .................................................................. 63 2.2.9 cDNA synthesis ....................................................................... 64 2.2.10 Polymerase Chain Reaction (PCR) ........................................ 64 2.2.11 Agarose gel electrophoresis .................................................. 67 2.2.12 Polyacrylamide Gel Electrophoresis (PAGE) ......................... 68 2.2.13 miRNA microarray design ...................................................... 69 2.2.14 miRNA microarray hybridization ............................................ 70 2.2.15 miRNA Quantitative Real Time Polymerase Chain Reaction (QRT-PCR) ......................................................................................
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