UUnniivveerrssiittyy ooff LLoouuiissvviillllee TThhiinnkkIIRR:: TThhee UUnniivveerrssiittyy ooff LLoouuiissvviillllee''ss IInnssttiittuuttiioonnaall RReeppoossiittoorryy Electronic Theses and Dissertations 8-2017 MMiiRRNNAA eexxpprreessssiioonn cchhaannggeess iinn aarrsseenniicc--iinndduucceedd sskkiinn ccaanncceerr iinn vviittrroo aanndd iinn vviivvoo.. Laila Al-Eryani University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Medicine and Health Sciences Commons RReeccoommmmeennddeedd CCiittaattiioonn Al-Eryani, Laila, "MiRNA expression changes in arsenic-induced skin cancer in vitro and in vivo." (2017). Electronic Theses and Dissertations. Paper 2767. https://doi.org/10.18297/etd/2767 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. MIRNA EXPRESSION CHANGES IN ARSENIC-INDUCED SKIN CANCER IN VITRO AND IN VIVO By Laila Al-Eryani A Dissertation Submitted To The Faculty of the School Of Medicine of the University Of Louisville In Partial Fulfillment of the Requirements For The Degree Of Doctor of Philosophy in Pharmacology and Toxicology Department Of Pharmacology and Toxicology University Of Louisville Louisville, Kentucky August 2017 MIRNA EXPRESSION CHANGES IN ARSENIC-INDUCED SKIN CANCER IN VITRO AND IN VIVO By Laila Al-Eryani Dissertation approved on August 03, 2017 By the following Dissertation Committee: J. Christopher States, Ph.D. Carolyn M. Klinge Ph.D. Shesh N. Rai, Ph.D. Chendil Damodaran, Ph.D. Theodore Kalbfleisch, Ph.D. ii ACKNOWLEDGMENTS I would like to express my deepest gratitude to my mentor Dr. J. Christopher States for his persistent help, guidance, motivation, patience and very big and kind heart through my Ph.D. journey. I want also to thank my committee members for their guidance, support and assistance starting form Dr. Damodaran and his lab members and Dr. Tyagi and Dr. Pal for all their technical help. I would also like to thank Dr. Shesh Rai and Dr. Pan for running the statistical analyses. I want also to thank Dr. Kalbfleisch for his help with the NGS data and Dr. Klinge for her advice and helping with troubleshooting. I also sincerely thank the DNA Core manager at the University of Louisville, Sabine Waigel, for all the tremendous help with hybridization microarray analyses and other analyses and being always welcoming and welling to help. Special thank you goes to Vennila Arumugam for all the technical support. My gratitude also goes to members of the Price Institute of Surgery at the University of Louisville, in particular Dr. Galandiuk, M. Robert Eichenberger, Dr. Chien, Dr. Sarojini for the access to instruments and enormous technical help for the LCM and skin lesions study. I would like to thank Dr. Malone for her tremendous help for diagnosing the skin lesions. I sincerely thank Dr. Jala for his technical help with the cell cycle assays and Josiah Hardesty for his technical help with western blot troubleshooting. iii I want also to thank Vanessa States for running the skin lesions preliminary miRNA expression data and Gregory States for designing the program used to facilitate measuring the cells migration. I wish also to thank all the summer students who contributed to the project, Sam Jenkins, who helped tremendously with the LCM, and Jana Peremarti. I also sincerely thank the Fulbright program for giving me the chance to pursue my graduate studies. Without their initial support during my master, I would have been able to pursue my Ph.D. Many thanks to the Integrated Program in Biomedical Sciences (IPIBS) and the department of Pharmacology and Toxicology for supporting me throughout my Ph.D. Last but not least I am very thankful to my amazing family, mom, dad and lamia along with my friends Banrida, Sabrin, Safa, Heather, Linda and Holly for all their continuous support which without I would never be where I am now. This work was supported in part by NIH grants R21ES023627, R01ES011314, T35ES014559, R25CA134283, and a Competitive Enhancement Grant from the University of Louisville Office of the Executive Vice President for Research and Innovation. Finally, I dedicate this dissertation to the memory of a beautiful soul and a very special person that left us too soon, my bother, Luai Al-Eryani. I will always love you, remember you and cherished you and every moment we spent together, you are deeply missed! iv ABSTRACT MIRNA EXPRESSION CHANGES IN ARSENIC-INDUCED SKIN CANCER IN VITRO AND IN VIVO Laila Al-Eryani August 03, 2017 Arsenic is a naturally prevalent metalloid. Chronic arsenic ingestion through drinking water causes skin cancer. Arsenic-induced cancer mechanisms are not well defined. Epigenetic changes, including microRNA expression changes, might be playing a role. This dissertation investigates the impact of miRNA expression changes in arsenic-induced skin cancer. MiRNA expression was measure and compared using 3 different techniques, RTq-PCR, hybridization arrays and RNA-sequencing. MiRNAs differential expression in skin lesions was phenotype- and stage-related. Immortalized human keratinocytes (HaCaT) were transformed by chronic low arsenite exposure serving as a model for arsenic-induced skin carcinogenesis. Early changes in miRNAs and target mRNAs contribute to arsenic-induced carcinogenesis. Throughout the time course of arsenic exposure, dysregulation of cells’ growth and cancer-related pathways were identified. Comparisons between the miRNA profiles in lesions and cells predict some miRNAs may serve as biomarkers and/or therapeutic targets for arsenic-induced tumors. This dissertation provides strong evidences of epigenetic changes related to carcinogenesis in arsenic-induced skin cancer. v TABLE OF CONTENTS Acknowledgements.…………………………………………………...………….. iii Abstract………………………...……………………………………………….… v List of Tables……………..…………………………………………………….… viii List of Figures…………………………………………………………….……… ix Chapter 1: General Introduction……………………………………………… 1 Chapter 2: MiRNA Expression Profiles of Premalignant and Malignant Arsenic-Induced Skin Lesions Introduction…………………………………………………….….. 13 . Materials and Methods…………………………………………… 19 Results…………………………………………………….……….. 21 Discussion.…………………………………………………….…... 33 Chapter 3: Differentially Expressed MiRNAs and MRNAs at Early Stages of Transformation of HaCaT Cells Chronically Exposed to Low Arsenite Introduction…………………………………………………….….. 40 Materials and Methods…………………………………………… 43 Results…………………………………………………….……….. 47 Discussion………….……………………………………………… 61 Chapter 4: Cell Cycle Pathway Dysregulation in Human Keratinocytes at Early Stages of Chronic Exposure to Low Arsenite Introduction…………………………………………………….….. 68 Materials and Methods…………………………………………… 70 Results…………………………………………………….……….. 73 Discussion………….……………………………………………… 78 vi Chapter 5: A Longitudinal Study on Small RNA And MRNA Expression Profiles in The Chronic Exposure HaCaT Model Introduction…………………………………………………….….. 80 Materials and Methods…………………………………………… 83 Results…………………………………………………….……….. 87 Discussion………….……………………………………………… 109 Chapter 6: MiRNA Expression Profiles Comparisons Across Three Different Techniques Introduction…………………………………………………….….. 112 Materials and Methods…………………………………………… 114 Results…………………………………………………….……….. 116 Discussion………….……………………………………………… 126 Chapter 7: Overall Discussion, Conclusions and Future Directions .……. 129 References……………………………………...…………………………….….. 135 Appendix: List of Abbreviations……………………………………………… 171 Curriculum Vitae……………………………………………...………………….. 174 vii LIST OF TABLES Table 2.1. Demographics of study population 22 Table 2.2. Differentially expressed miRNAs in Arsenic-induced 27 Skin Lesions and Their Expression Table 3.1. Differentially expressed small RNAs at both 3 and 7 52 weeks of exposure to arsenite Table 3.2. Top 10 pathways of the differentially expressed 38 54 mRNAs at both 3 and 7 weeks that are predicted targets of miRNAs differentially expressed at both 3 and 7 weeks Table 4.1. Pathways populated by differentially expressed mRNAs 75 7 weeks Table 5.1. MiRNAs differentially expressed at more than one time 88 point Table 5.2. Pathway analysis of differentially expressed genes at 7, 90 19 and 28 weeks Table 5.3. Pathway analysis of differentially expressed mRNAs 104 targets of differentially expressed miRNAs Table 6.1. Differentially expressed miRNAs at 3 weeks of 117 chronically arsenic- exposed/unexposed HaCaT cells using RT-qPCR array cards Table 6.2. Differentially expressed miRNAs at 3 weeks in exposed 119 cells vs. unexposed cells to those of unexposed cell vs. skin lesions Table 6.3. Differentially expressed miRNAs at 3 weeks of 121 chronically arsenic-exposed HaCaT cells in both hybridization microarrays and RT-qPCR array cards Table 6.4. Differentially expressed miRNAs at 7 weeks of 123 chronically arsenic-exposed HaCaT cells in both hybridization microarrays and Next-Generation Sequencing (NGS) Table 6.5. MiRNAs differentially expressed in chronically arsenic- 125 exposed HaCaT cells and arsenic-induced skin lesions viii