“Using liver miRNA profiles to predict chemical hepatocarcinogenesis” By Costas Koufaris A thesis submitted to Imperial College London for the degree of Doctor of Philosophy Department of Surgery and Cancer Biomolecular Medicine Imperial College London April- 12011 - ABSTRACT Industrial, agricultural, and pharmaceutical requirements drive the development of a plethora of new chemical entities each year, many of which -for example drugs, pesticides, and food additives- have to be assessed for potential human health hazard. The current benchmark for risk assessement is the lifetime rodent bioassay which is expensive, time-consuming, laborious, requires the sacrifice of numerous animals, and is often irrelevant to humans. Hence alternative strategies to the rodent lifetime bioassay for prediction of chemical carcinogens are being pursued, especially for the liver which is an organ frequently affected by exogenous chemicals due to its detoxifying and metabolic roles. Numerous studies in recent years support the important role of microRNAs in cancer development, including hepatocellular carcinoma. The principal hypothesis of this project was that hepatic microRNA signatures can contribute to the earlier prediction of chemical hepatocarcinogens. Examination of livers from male Fischer rats treated with six chemical hepatocarcinogens, with diverse mode of actions for 90 days revealed that all the tested hepatocarcinogens affected the liver miRNAome from that early stage. Interestingly, a small set of microRNAs were identified whose expression was frequently deregulated by the hepatocarcinogens. Bioinformatic analysis indicates that these microRNAs can regulate pathways which are important in hepatocellular carcinoma. A more detailed investigation of one of those hepatocarcinogens, phenobarbital, showed that its effects on liver microRNAs were both dose and time dependent, with a progressive induction of specific microRNA clusters.Thus this study was the first to investigate in depth the effects of chemical hepatocarcinogens on the liver miRNAome and supports the potential usefulness of hepatic microRNA signatures in risk assessment. - 2 - Statement of Originality I affirm that all the work presented in this thesis is my own original work, unless otherwise stated. Where the work of others has been used, it has been dully acknowledged. Costas Koufaris April 2011 - 3 - “Σα βγεις ζηον πηγαιμό για ηην Ιθάκη, να εύχεζαι νάναι μακρύς ο δρόμος, γεμάηος περιπέηειες, γεμάηος γνώζεις…” ”As you set out on the way to Ithaca, wish that the road is a long one, full of adventures, full of knowledge...” Constantine P. Cavafy - 4 - Dedication To my parents Acknowledgments Like Odysseus’s voyage to Ithaca, the road to completing a PhD can be a long one, but also full of adventures and knowledge. First I would like to express my gratitude to my supervisor, Professor Nigel J. Gooderham, without whom I may never have set out on this journey. Throughout the last three and a half years he has always been available to offer his advice and guidance. I would particularly want to thank him for instilling in me the confidence that is required for pursuing a PhD. I have thoroughly enjoyed and I am proud to have been his student and a member of his lab. I would also like to acknowledge Dr. Jayne Wright and Dr.Richard A. Currie for their great effort, assistance, and encouragement. This project would have been much weaker without their help. I am also thankful to the BBSRC for funding my PhD. I have also greatly appreciated all the assistance, advice, encouragement, and companionship that I have received from the various members of my lab during my PhD: Mihalis Papaioannou, Corrinne Segal, Reshat Reshat, Qianxin Wu, Nurul Huda Abd Karim, Kuan-wei Chen, Dr. William Edmands, Dr. Rhiannon David, and Dr. Nor Aini Saidin. Also my gratitude to R.F. for being a source of inspiration. I sincerely wish them all the best in the future. I also appreciate the numerous other people (staff and students) who have aided me in various circumstances. Last but not least, I am grateful to Vicky for being my safe harbour, my guiding star, and a gentle breeze driving me on during this journey. - 5 - Contents List of Figures……………………………………………..……..…12 List of Tables……………………………………………………..…16 List of abbreviations……………………………………………... 19 Chapter 1- Introduction……………………………………..23 1.1 Mechanisms of carcinogenesis……………………………….…….23 1.1.1 Carcinogenesis is a multi-step, progressive process………….…..…...24 1.1.2 Genetic and epigenetic mechanisms of gene disruption in cancer…….30 1.1.3 Mechanisms of carcinogenesis by exogenous chemicals…………...…34 1.2 MicroRNAs have important roles in cancer…………………….......42 1.2.1 Non-coding RNAs are conserved in eukaryotes…………………….....42 1.2.2 Biogenesis and function of mature miRNAs…….…………………….43 1.2.3 MicroRNAs in cancer: small but deadly………………………………49 1.3. Liver is a frequent target of chemical carcinogens ……………..….53 1.3.1. Liver architecture and function…………………………………..……53 1.3.2. Aetiology, types, and frequencies of liver cancers……………………57 1.3.3 Phenobarbital is a prototype of non-genotoxic hepatocarcinogens…....61 1.3.4 Involvement of miRNAs in hepatocellular carcinomas ……...…….….68 1.4. Cancer risk assessment strategies…………………………………..70 1.4.1. Rodent lifetime bioassays are the benchmark for risk assessment…....70 - 6 - 1.4.2 Alternative strategies to the rodent lifetime bioassays……………......73 1.5 Justification, hypothesis, and objectives………………….........……75 Chapter 2- Materials and Methods……………...........…..…78 2.1 Chemicals and reagents used………………………...…………..….79 2.2 Animal studies……………………………………………...…...…..80 2.2.1 Tested compounds ……………………………………………....….…80 2.2.2 Design of animal studies………………………………………….....…81 2.3 MicroRNA microarrays……………………………………….…….84 2.3.1 RNA extraction…………………………………………………….…..84 2.3.2 Assessment of RNA quantity, purity and integrity……………….……85 2.3.3 RNA labelling, hybridisation, and scanning………………..........…….86 2.3.4 Pre-processing and normalisation of miRNA microarray data….......…89 2.4 Polymerase chain reaction (PCR)……………………………...........91 2.4.1 Real-time Quantitative PCR (qPCR)……… …………….................…91 2.4.2 Semi-quantitative PCR………………………………………………...93 2.5 Immunoblotting……………………………………………….…….95 2.5.1 Protein extraction from liver samples .......................………….........…95 2.5.2 Protein detection and quantification………………………...….…..….96 2.6 Methylation analysis……………………………………...….…..….97 2.6.1 DNA extraction and quantification………………….......................…..97 2.6.2 Global DNA methylation analysis………………….............…..…..….98 - 7 - 2.7 RLM-RACE...............................................................................….98 2.8 In silico analysis……………… .................................…………..100 2.9 Clustering and classification of samples……………..………….101 2.9.1 Hierarchical clustering………………………….....................…….101 2.9.2 Sample classification……………………………………….............102 2.10 Statistical analysis…………………………………………....…102 Chapter 3-Effects of 90 day treatment with various............103 hepatocarcinogenic and non-hepatocarcinogenic compounds on the liver microRNAome 3.1. Introduction...……………………….………........………………104 3.2. Effects of chemical treatments on animals..………….......………107 3.3. Evaluation of quality and processing of microarray data...............110 3.3.1 PCA and Quality Control metrics of samples......................................110 3.3.2 Filtering of miRNA data.......................................................................114 3.3.3. Comparison of microarray technical replicates...................................117 3.4 Deregulation of hepatic miRNAs by hepatocarcinogens.................119 3.4.1 MiRNA profiles are distinct for carcinogen-treated livers...................119 3.4.2 Identification of possible miRNA biomarkers of carcinogenicity.......124 3.4.3 MiRNA profiles are associated with the MOA of chemicals...............133 3.4.4 Comparison of chemical effects on mRNAs and miRNAs.................134 - 8 - 3.4.5 Correlation of “biomarker” miRNAs with hepatic parameters............136 3.5 Chemical-specific miRNA profiles..................................................137 3.5.1 PB-type compounds have characteristic miRNA profiles....................137 3.5.2. Effects of genotoxic 2-AAF on microRNAs.......................................140 3.5.3 DEHP shows some similarities to PB-type compounds......................143 3.5.4. MiRNA profiles of MP HCL, MON, and Chl.Ac...............................145 3.6 qPCR validation of miRNA changes...............................................146 3.7 Discussion........................................................................................147 Chapter 4- Hepatic microRNA profiling of rats........................152 treated with dietary phenobarbital 4.1 Introduction......................................................................................153 4.2 Effects of chemical treatments on animals...................................... 155 4.3 Quality control of microarray data...................................................158 4.4 Clustering Analysis of samples........................................................164 4.5 Identifying differentially expressed miRNAs..................................171 4.5.1 MiRNAs deregulated by mitogenic PB doses.....................................171 4.5.2 MiRNAs deregulated by carcinogenic 1000 ppm PB dose.................174 4.5.3 Progressive induction of miR-200b and miR-96 from 14 to