University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Nutrition & Health Sciences Dissertations & Theses Nutrition and Health Sciences, Department of 4-2015 MICRORNAs ARE ABSORBED IN BIOLOGICALLY MEANINGFUL AMOUNTS FROM NUTRITIONALLY RELEVANT DOSES OF COW’S MILK AND CHICKEN EGGS AND AFFECT GENE EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS, CELL CULTURES, AND MOUSE LIVERS Scott Baier University of Nebraska-Lincoln, [email protected] Follow this and additional works at:http://digitalcommons.unl.edu/nutritiondiss Part of theFood Microbiology Commons, and theMolecular, Genetic, and Biochemical Nutrition Commons Baier, Scott, "MICRORNAs ARE ABSORBED IN BIOLOGICALLY MEANINGFUL AMOUNTS FROM NUTRITIONALLY RELEVANT DOSES OF COW’S MILK AND CHICKEN EGGS AND AFFECT GENE EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS, CELL CULTURES, AND MOUSE LIVERS" (2015).Nutrition & Health Sciences Dissertations & Theses. 50. http://digitalcommons.unl.edu/nutritiondiss/50 This Article is brought to you for free and open access by the Nutrition and Health Sciences, Department of at DigitalCommons@University of Nebraska - Lincoln. 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MICRORNAs ARE ABSORBED IN BIOLOGICALLY MEANINGFUL AMOUNTS FROM NUTRITIONALLY RELEVANT DOSES OF COW’S MILK AND CHICKEN EGGS AND AFFECT GENE EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS, CELL CULTURES, AND MOUSE LIVERS by Scott Baier A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Interdepartmental Area of Nutrition Under the Supervision of Professor Janos Zempleni Lincoln, Nebraska April, 2015 MICRORNAs ARE ABSORBED IN BIOLOGICALLY MEANINGFUL AMOUNTS FROM NUTRITIONALLY RELEVANT DOSES OF COW’S MILK AND CHICKEN EGGS AND AFFECT GENE EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS, CELL CULTURES, AND MOUSE LIVERS Scott Baier, Ph.D. University of Nebraska, 2015 Advisor: Janos Zempleni Throughout the twenty-first century, evidence has been continually increasing to show the importance of epigenetic regulation in health. While the term “epigenetics” can be applied to many different processes, the focus of this dissertation will be on microRNAs and chromatin structure. Ultimately, both of these forms of epigenetic regulation can be used to fine tune gene expression based on environmental cues. The first three chapters of the dissertation focus on microRNA bioavailability, stability, and function from two commonly consumed food products: cow’s milk and chicken eggs. This important work has been the first of its kind to demonstrate the bioavailability and function of dietary, animal-based microRNAs. In order to test our hypothesis that dietary microRNA are indeed bioavailable, many experimental protocols have been used. Human feeding studies are the prominent tool used, but important discoveries have been made in cell culture and in a mouse feeding study that add to the significance of this work. The final chapter will focus on a well-known histone modifier: sulforaphane. There have been many positive roles attributed to this compound, but the work presented here describes some potentially negative consequences of high sulforaphane intake. Overall, the conclusion of this work is dietary microRNA are bioavailable and can regulate endogenous gene expression. For the scientific field to determine the contribution of dietary microRNAs to overall health, much more work will need to be performed but the evidence provided in this dissertation indicates it is likely dietary microRNAs play a key role in overall epigenetic regulation. Ongoing studies in our laboratory to further this work and answer some of the current questions are also briefly described. iv ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. Janos Zempleni for providing me an opportunity to obtain my doctoral training in his laboratory. Dr. Zempleni not only taught me valuable laboratory skills for use in conducting experiments, but I learned even more from him in how to think like a scientist and critically review and plan scientific work. I also want to extend appreciation to my committee members for their valuable contribution to my dissertation work: Dr. Bin Yu, Dr. Regis Moreau, and Dr. Vicki Schlegel. In addition to the important role Dr. Zempleni played in my training, many others were crucial for my growth as a researcher. Without a doubt, the most of important of those is a former postdoctoral scientist from our lab, Samudra Wijeratne. When I initially came to work in the lab, Samudra helped teach me many procedures and was always willing to troubleshoot experiments along the way. Other individuals that have helped me in different ways and are in need of recognition are Jing Xue, Mengna Xia, Dandan Liu, Wei Kay Eng, David Giraud, Daniel Câmara Teixeira, Elizabeth Cordonier, Tovah Wolf, Katherine Howard, Rio Jati Kasumi, Dr. Jennifer Wood, and Fang Xie. While the focus is often on the research done in the lab, there is equally important work done to make sure everything else can run smoothly. Many people are responsible for making this happen, probably more than I even know, but some members of the Nutrition and Health Science and CEHS staff have been especially important and I would like to thank Donna Hahn, Lori Rausch, Jolene Walker, Carrie Brownyard, Connie Wieser, Lori Beals, Ann Grasmick, and Sarah Gibson. I also would like to thank our Department Chair, Dr. Timothy Carr, for his advice and guidance throughout my program. v Finally, saving the most important for last, I want to thank my wife, Ashley, for her unending support and love. I owe my success to her and my parents, without whom this work would not have been possible. Scott Baier April, 2015 vi TABLE OF CONTENTS LIST OF FIGURES vi LIST OF TABLES ix INTRODUCTION 1 Reference 9 AUTHOR CONTRIBUTIONS 14 CHAPTER I. 15 Reference 33 CHAPTER II. 47 Reference 60 CHAPTER III. 64 Reference 74 CHAPTER IV. 84 Reference 95 OUTLOOK 106 vii LIST OF FIGURES INTRODUCTION Figure 1. Biogenesis and function of microRNA. 2 Figure 2. Exosome secretion, stability, and uptake. 4 Figure 3. Modeling of a sulforaphane/HDAC interaction. 6 Figure 4. Retrotransposon translocates between different genomic loci. 7 CHAPTER I Figure 1. Plasma time curves of microRNA following milk consumption. 40 Figure 2. PBMC miRNA abundance after milk consumption. 41 Figure 3. Effects of milk microRNAs on gene expression in humans. 42 Supplemental Figure 1. Schematic of plasmids used in this study. 44 Supplemental Figure 2. Time courses of PBMC gene expression 45 CHAPTER II Figure 1. Plasma time curves of miR-181b following an egg meal. 57 Figure 2. Erythrocyte miRNA abundance following egg consumption. 58 Figure 3. Effects of egg consumption on PBMC gene expression. 59 viii CHAPTER III Figure 1. Loss of miRNA during milk pasteurization and homogenization. 80 Figure 2. Effect of storage on miRNA concentrations. 81 Figure 3. Effect of heating on miRNA concentration. 82 CHAPTER IV Figure 1. Effect of SFN on LTR transcription in IMR-90 cell cultures. 101 Figure 2. Effect of SFN on p21 transcription in IMR-90 fibroblast cultures. 102 Figure 3. AUC for LTR and p21 expression increases following three broccoli sprout doses in healthy adults. 103 Figure 4. Effect of broccoli sprout consumption on PBMC enrichment of H3K9ac mark in LTR 15. 104 ix LIST OF TABLES CHAPTER I Table 1. Pharmacokinetics analysis of plasma time curves of microRNAs after milk meals in healthy adults. 39 Supplemental Table 1. List of primers used in this study. 43 Supplemental Table 2. Composition of diets used in mouse feeding study. 46 CHAPTER II Table 1. Pharmacokinetics analysis of plasma time curves of miR-181b after egg meals in healthy adults. 56 CHAPTER III Table 1. Concentration of miRNAs-29b and -200c in dairy products 83