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UNDERSTANDING SHARED PATHOGENESIS BETWEEN CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) AND LUNG CANCER BY MEANS OF CELL SPECIFIC GENOMICS CLARA EMILY GREEN A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY The Institute of Inflammation and Ageing College of Medical and Dental Sciences University of Birmingham February 2018 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Introduction COPD (Chronic Obstructive Pulmonary Disease) and lung cancer are related conditions associated with inflammation. Relatively little focus has been given to the endothelium, through which inflammatory cells transmigrate to reach the lung. We sought to determine if coding and non-coding alterations in pulmonary endothelium exist in COPD and lung cancer. Methods Patients with and without COPD undergoing thoracic surgery were recruited. Pulmonary Endothelial Cells were isolated from lung and tumour and extracted RNA (ribonucleic acid) used for miRNA (micro-RNA) and mRNA (messenger RNA) microarrays. Ingenuity pathway analysis (IPA) was also carried out. Results 2071 genes and 43 miRNAs were significantly upregulated in COPD. 4 targets were validated by quantitative polymerase chain reaction, of which miR-181b-3p was chosen for functional validation. Another target, miR-429, was also increased in lung tumour. Several cancer-related pathways such as transforming growth factor- β were altered in the IPA. There was significantly reduced tube formation and endothelial sprouting in Human umbilical vein endothelial cells transfected with miR-181b-3p, consistent with an effect on angiogenesis. Conclusions Upregulation of miR-181b-3p reduces tube formation and sprouting by endothelial cells. This might be significant in the development of emphysema as lung vasculature is important in the structural maintenance of alveoli. Dedication I dedicate this thesis to my husband Robert Green for all of your encouragement and support. Acknowledgements Firstly I would like to thank my supervisors Dr. Alice Turner and Prof. Roy Bicknell for the invaluable advice and support you have given me throughout the past four years. It has been a pleasure to work with both of you. I am also grateful for the help and assistance of members of both the Respiratory and Bicknell/Heath groups without whom I would have been unable to complete the necessary laboratory work. Thank you to all of the staff responsible for the managing of, and collection of tissue for, the Midlands Lung Tissue Consortium, including Prof. David Thickett, Prof. Babu Naidu, Dr. Gerald Langman, Thoracic surgeons and Thoracic research nurses at Birmingham Heartlands Hospital. I am also grateful to Dr. Zsuzsanna Nagy for her advice regarding microarray analysis and for the use of Ingenuity Pathway Analysis. Finally, I would like to thank my family for their endless help and support throughout the course of this study. Thank you to my husband Rob for listening and providing advice throughout my degree. Thank you to my sister Elena who provided me with support over the phone. Thank you to my parents John and Olivia for providing me with hours of childcare so I could complete this work. Thank you to my sons Eric and Arthur for providing me with much needed love and joy during the course of study. Table of Contents Chapter One Introduction ……………………………………………………………………… 1 1.1 Chronic obstructive Pulmonary Disease (COPD)………………………... 2 1.1.1 Clinical Features…………………………………………………… 3 1.1.2 Management……………………………………………………….. 9 1.1.3 Pathogenesis………………………………………………………. 12 1.2 Lung Cancer………………………………………………………………….. 16 1.2.1 Clinical Features…………………………………………………… 16 1.2.2 Management……………………………………………………….. 19 1.2.3 Pathogenesis………………………………………………………. 21 1.3 Evidence for the shared pathogenesis of COPD and lung cancer……... 26 1.4 The role of the endothelium in the lung…………………………………….28 1.4.1 The role of the endothelium in COPD…………………………… 34 1.4.2 The role of the endothelium in lung cancer…………………….. 39 1.5 The study of micro-RNA and messenger RNA expression in the pathogenesis of lung disease………………………….……………………...…41 1.5.1 The study of micro-RNAs in lung disease………………………..41 1.5.2 The study of messenger RNAs in lung disease…………………50 1.5.3 Pathway analysis of genomic data in the setting of lung cancer and COPD………………………………………………………………….53 1.6 Isolation of Human Pulmonary Endothelial Cells (HPECs) for culture….57 1.7 Hypotheses of this thesis…………………………………………………….59 1.8 Aims…………………………………………………………………………… 60 Chapter Two Methods…………………………………………………………………………… 63 2.1 Patient data collection………………………………………………………. 64 2.1.1 Patient selection……………………………………………........... 64 2.1.2 Clinical data………………………………………………………… 64 2.2 Statistical analysis of clinical data…………………………………………. 67 2.3 Collection of lung tissue…………………………………………………….. 67 2.3.1 Extraction of macrophages……………………………………….. 67 2.3.2 Extraction of endothelium…………………………………........... 70 2.4 Preparation of samples for microarray analysis………………………….. 73 2.4.1 RNA Extraction…………………………………………………….. 73 2.4.2 Quality control of RNA prior to microarray analysis……………. 75 2.4.3 Labelling RNA for mRNA microarray analysis…………………. 75 2.4.4 Hybridisation of mRNA for microarray…………………………... 78 2.5 Microarray Analysis………………………………………………………….. 81 2.5.1 Quality analysis of microarray results……………………........... 81 2.5.2 Assessment of differential gene expression using Limma……..86 2.5.3 Assessment of false discovery rate (FDR)……………………… 89 2.5.4 Assessment of differential gene expression using Significance Analysis of Microarrays (SAM)…………………………………………..90 2.5.5 Ingenuity Pathway Analysis (IPA)……………………………….. 92 2.6 Reverse transcription………………………………………………………... 97 2.6.1 Reverse transcription of RNA to cDNA for RNA samples of concentration >300 ng/µl………………………………………………... 97 2.6.2 Reverse transcription of RNA to cDNA for RNA samples of concentration <300 ng/µl………………………………………………... 97 2.6.3 Reverse transcription of miRNA…………………………………. 98 2.7 Real-time Polymerase chain reaction (qPCR)……………………………. 98 2.7.1 qPCR of mRNA……………………………………………………. 98 2.7.2 qPCR of miRNA…………………………………………………… 99 2.7.3 Analysis of qPCR results………………………………………….101 2.8 Culture of Mouse Lung Endothelial Cells (MLECs)………………………103 2.8.1 Collagenase digestion of lung tissue…………………………….103 2.8.2 Negative selection of macrophages using magnetic beads…...104 2.8.3 Positive selection of endothelial cells by magnetic beads…….. 106 2.9 Culture of Human Pulmonary Endothelial Cells (HPECs)………………..108 2.9.1 Collagenase digestion of lung tissue……………………………. 108 2.9.2 Positive selection of endothelial cells by magnetic beads……..110 2.10 Cell Storage………………………………………………………………….111 2.11 Flow cytometry……………………………………………………………… 111 2.11.1 Flow cytometry to look for CD31 expression in cells isolated from mouse lung……………………………………………………………….. 111 2.11.2 Flow cytometry to look for CD31 expression in cells isolated from the lung……………………………………………………………………. 113 2.11.3 Flow cytometry for cell cycle analysis……………………….….115 2.12 Cell culture for cellular functional work……………………………………120 2.13 miRNA Inhibition and Overexpression…………………………………… 120 2.13.1: Transfection of a miRNA inhibitor or mimic into HUVEC in a 6 well plate…………………………………………………………………...120 2.14 Cell growth assay……………………………………………………………125 2.15 Matrigel tube formation assay……………………………………………..125 2.16 Scratch wound assay……………………………………………………....126 2.17 Spheroid assay……………………………………………………………...129 2.17.1 Preparation of the spheroid assay………………………….......129 2.17.2 Analysis of spheroids using ImageJ…………………………….131 Chapter Three MicroRNA and Messenger RNA expression in lung endothelium………133 3.1 Hypothesis…………………………………………………………………….134 3.2 Aims of this chapter…………………………………………………………..134 3.3 miRNA expression in the lung endothelium………………………………. 134 3.3.1 Subjects…………………………………………………………….. 134 3.3.2 Combined analysis of 2014 and 2015 microarrays……………. 134 3.3.3 Computer prediction of targets of identified miRNAs………….. 140 3.3.4 qPCR validation of miRNA targets ………………………………144 3.3.5 Validation of potential miRNA targets using qPCR in lung cancer ……………………………………………………………………………... 147 3.4 mRNA expression in the lung endothelium……………………………….. 147 3.4.1 Subjects…………………………………………………………….. 147 3.4.2 Combined analysis of 2014 and 2016 microarrays……………. 147 3.4.3 Tumour mRNA analysis…………………………………………... 156 3.4.4 Validation of potential mRNA targets using qPCR…………….. 159 3.5 Discussion……………………………………………………………………. 163 3.5.1 miRNA expression in pulmonary endothelium…………………. 163 3.5.2 mRNA expression in pulmonary endothelium………………….. 170 3.5.3 Limitations………………………………………………………….. 174 3.6 Conclusions…………………………………………………………………... 175 Chapter Four Pathway analysis of microRNA and messenger RNA expression data…………………………………………………………………………………176 4.1 Hypothesis…………………………………………………………………….177 4.2 Aims of this chapter…………………………………………………………. 177 4.3 Introduction…………………………………………………………………… 177 4.4 Results……….……………………………………………………………...... 177 4.4.1 Diseases and biological
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