COMPUTATIONAL ANALYSIS OF GENE EXPRESSION IN COMPLEX DISEASE A Thesis Presented to The Academic Faculty by Andrew Douglas Huang In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Bioinformatics in the School of Biology Georgia Institute of Technology December 2014 COPYRIGHT © ANDREW DOUGLAS HUANG 2014 COMPUTATIONAL ANALYSIS OF GENE EXPRESSION IN COMPLEX DISEASE Approved by: Dr. John F. McDonald, Advisor Dr. Kathyrn M. Momary School of Biology Department of Pharmacy Practice Georgia Institute of Technology Mercer University Dr. Nathan Bowen Dr. Jung H. Choi Department of Biological Science School of Biology Clark Atlanta University Georgia Institute of Technology Dr. George W. Daneker School of Biology Georgia Institute of Technology Date Approved: August 5th, 2014 For my parents, who share my dream. ACKNOWLEDGEMENTS The work described herein would have been impossible without significant efforts in the wet side of the McDonald lab from L. DeEtte Walker, Lilya Matyunina, Lijuan Wang, Laura Kapa, Jing Chen, GT Wagner, Ken Scarberry, and Shubin Shahab, as well as their scientific insights. I would also like to thank my friends on the dry (and sometimes wet) side of the McDonald lab: Roman Mezencev, Neda Jabbari, Chris Hill, Vinay Mittal, Loukia Lili, and Gaurav Arora for their ongoing discussions and motivation. The support of all in the McDonald lab, especially from John F. McDonald, helped propel me through this convoluted pathway to my doctorate. The work on firefighter coronary health and drug-eluting stents would not have been possible without the hard work of our collaborators at St. Joseph’s Hospital. In particular I’d like to thank Radhika Gadesam, Cathy Skrifvars, Lakshmana Pendyala, Angela Stoppar, Dongming Hou, Kumar Natarajan, as well as Kathryn Momary of Mercer University, for their work gathering and interpreting data. Particular recognition goes to Robert Superko and Jaipal Singh for spearheading these projects and providing insights into the nature of collaboration. For their support in my as part of Stephen Harvey’s lab, I’d like to thank Tom Caulfield, Batsal Devkota, M. Burak Boz, Anton Petrov, Amanda McCook, Marcella Aliste, Geoff Rollins, Minmin Pan, Yingying Zeng, and Piyush Ranjan. The support, friendship, and last minute paperwork filings of a number of Georgia Tech staff and faculty, including Todd Streelman, I King Jordan, Marc Pline, Kevin iv Roman, Lisa Redding, Nael McCarty, Inga Schmidt-Krey, Soojin Yi, Steve Harvey, Steven Kudravi, Rupal Cutting, Jennifer Leavey, Angie Lessard, Francesca Storici, Rob Butera, Laura Cook, Al Merrill, Chong Shin, Mirjana Brockett, Yuhong Fan, Roger Wartell, and Verene Lancaster, kept me going through these years. I also wanted to recognize the fine fellowship of many from around Georgia Tech, some of whom I’d met my first year and others gathered along the way, including Nick Parnell, Jon Sylvester, Sara Delavan, Karla Vincent, Ahsan Huda, Lee Katz, Daudi Jjingo, Andrey Kislyuk, Jen Kovacs, Rachel Lasley, Sam Parks, Amin Momin, Elizabeth Padilla, Randi Wilson, Jason Landrum, Taylor Updegrove, Wenhan Zhu, Jittima Piriyapongsa, Vardges Ter-Hovhannisyan, Adam Offenbacher, Eric Dammer, Aarti Urs, Jan Davidson-Drexel, Jianrong Wang, Wendy Morrison, Diya Mohan, Chiao Hsiao, Christine Fennessy, Brandy Jones, Astrid Layton, Andrew Farmer, Sean Kolk, Austin Ward, Ikay Okafor, Joan Fernandez, Vanessa Smet, Marissa Meier, Mariel Frieberg, Steve Heitner, Beth Kelly, Chris Kelly, Rhadika Dhingra, Miller Templeton, David Knobbe, Matt Marcus, Will Reeves, Bartek Ilkowski, Brandon Strellis, Heather Holmes, Zach Marion, Nikolai Curtis, Luke Beardslee, Henrik Muhe, Edouard De Watteville, Giuseppe Trianiti, and many others. Outside Georgia Tech, I’d also like to recognize Paige Leitman, Ben Heisler, Erik Hofmeister, David Brainard, Gillen Heisler, Ron Franke, Joe Streaky, Mike Hanftt, Alex Medina, Sarah Peck, Ken Blumreich, Amanda Loftis, Chris Russell, Ignacio Portabella, and Jessica Halpin for keeping me grounded. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iii LIST OF TABLES viii LIST OF FIGURES x SUMMARY xi CHAPTER 1 Introduction 1 1.1 Gene expression 1 1.2 Ovarian cancer 7 1.3 Atherosclerosis 12 1.4 References 17 2 Improving Framingham risk score estimation of coronary calcification and carotid intima-medial thickness in firefighters 28 2.1 Introduction 28 2.2 Methods 31 2.3 Results 35 2.4 Discussion 47 2.5 References 53 3 Inflammation and extracellular matrix genes differentially expressed in coronary arteries following zotarolimus- and sirolimus-eluting stent implantation 59 3.1 Introduction 59 3.2 Materials and methods 61 3.3 Results 65 3.4 Discussion 82 vi 3.5 Conclusion 84 3.6 References 84 4 Reversible expression of epithelial cell markers and phenotypes in ovarian cancer cells treated with miR-429 90 4.1 Introduction 90 4.2 Methods 95 4.3 Results 98 4.4 Discussion 133 4.5 References 135 5 Conclusion 145 5.1 Firefighter coronary risk factor study 145 5.2 Drug-eluting stent gene expressions study 146 5.3 miR-429 treatment of ovarian cancer cell study 149 5.4 References 152 VITA 154 vii LIST OF TABLES Page Table 2.1: Characteristics (means) of the sampled firefighter population 36 Table 2.2: Comparison of common characteristics between firefighters at different risk levels as determined by CAC 36 Table 2.3: Comparison of common characteristics between firefighters at different risk levels as determined by CIMT 36 Table 2.4: Low sensitivity for CAC at traditional FRS thresholds 41 Table 2.5: Low sensitivity for CIMT at traditional FRS thresholds 41 Table 2.6: Improved FRS correspondence to CAC at 6% risk threshold 45 Table 2.7: Improved FRS correspondence to CIMT at 8% risk threshold 45 Table 2.8: FRS smoking adjustment 46 Table 2.9: Changes in traditional FRS threshold correspondence with CAC with smoking adjustment 46 Table 2.10: Changes in traditional FRS threshold correspondence with CIMT with smoking adjustment 46 Table 2.11: Changes in FRS correspondence with CAC with smoking adjustment at 12% threshold 50 Table 2.12: Changes in FRS correspondence with CIMT with smoking adjustment at 20% threshold 50 Table 3.1: Analyzed stents and their implant positions 64 Table 3.2: Significantly differentially expressed genes 66 Table 3.3: Extracellular matrix process genes 71 Table 3.4: Potassium ion channel activity and ion channel activity genes differentially expressed in SES compared to BMS 76 Table 3.5: Extracellular matrix genes and basement membrane genes differentially expressed in ZES compared to SES 78 Table 3.6: Muscle component gene set genes differentially expressed in ZES compared to SES 80 viii Table 3.7: Inflammatory process genes differentially expressed in SES compared to BMS and ZES compared to BMS 80 Table 4.1: Multiple genes associated with EMT or MET are differentially expressed across the 144 hour time course 99 Table 4.2: Genes that do not return to their 0 hour expression levels by 144 hours post- transfection 104 Table 4.3: Genes targeted by miR-429, as predicted by miRanda, that are differentially expressed over the time course (0-144 hours) 113 Table 4.4: Selected genes differentially expressed in the miR-429 treated cells during the 0 to 48 hour time span 120 Table 4.5: Selected genes differentially expressed in the miR-429 treated cells during the 48 to 144 hour time span 124 Table 4.6: Genes associated with regulation of the epithelial-mesenchymal transition that are differentially expressed over the time course (0-144 hours) 126 ix LIST OF FIGURES Page Figure 2.1: Coronary artery calcification vs. Framingham Risk Score 38 Figure 2.2: Carotid intima-media thickness vs. Framingham Risk Score 39 Figure 2.3: Receiver-operator characteristic curve for Framingham Risk Score in predicting coronary artery calcification status 42 Figure 2.4: Receiver-operator characteristic curve for Framingham Risk Score in predicting carotid intima-media thickness status 43 Figure 2.5: Receiver-operator characteristic curve for Framingham Risk Score prediction of coronary calcification with smoking adjustment 48 Figure 2.6: Receiver-operator characteristic curve for Framingham Risk Score prediction of carotid intima-medial thickness with smoking adjustment 49 Figure 3.1: Hierarchical clustering of 3008 genes differentially expressed among arterial tissue exposed to bare metal, sirolimus-eluting, and zotarolimus-eluting stents 69 Figure 4.1: Clustered fold-change values of probes display a large change at 48 hours102 Figure 4.2: By 144 hours, most genes in miR-429 treated cells have the same average expression as at 0 hours 103 Figure 4.3: Fold-change over time of selected genes with significantly different expression at 144 hours than at 0 hours 106 Figure 4.4: miR-429 transfection in HEY cells induces morphological changes characteristic of MET and then EMT 110 Figure 4.5: Gene enrichment analysis of genes differentially expressed across the 0-144 hour time course 111 Figure 4.6: Gene set enrichment of genes differentially expressed in the miR-429 treated cells during the 0-48 and 48-144 hour time span 121 x SUMMARY Cardiovascular disease (CVD) causes 45% of on-duty firefighter fatalities, a high fraction even when compared to the risk of CVD found in other first-responder professions like police work and emergency medical services. Monitoring and managing firefighter cardiac health is important for both individual health and public safety. In our study, we are interested in assessing the utility of the most commonly used risk assessment scoring, known as the Framingham Risk Score, in evaluating the atherosclerotic risk in asymptomatic firefighters. To this end we determined FRS for 159 male firefighters from Gwinnett County, Georgia, and compared their risk categorization against their known atherosclerotic burden as determined by CIMT and CAC. While the 20% FRS threshold, corresponding to medium risk, had a high specificity for both CAC and CIMT, it also had a low sensitivity (17% and 40%, respectively), indicating that a large percentage of individuals with clinically significant atherosclerosis are being misclassified.