University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 1-23-2018 Functional Study of Long Noncoding RNA H19 in Muscle and Liver Na Zhang University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Zhang, Na, "Functional Study of Long Noncoding RNA H19 in Muscle and Liver" (2018). Doctoral Dissertations. 1705. https://opencommons.uconn.edu/dissertations/1705 Functional Study of Long Noncoding RNA H19 in Muscle and Liver Na Zhang, PhD University of Connecticut, 2018 Abstract The study in this thesis mainly focus on understanding the regulatory roles of LncRNA H19 in the muscle and liver. In order to better understand the role of H19 in muscle differentiation, we utilized RNA-seq to compare the differential gene expression between control and H19 knockdown cells during C2C12 myoblast differentiation. This study can lead us to identify candidate genes that are regulated by H19 during muscle differentiation. We also found that H19 plays an important role in the liver through regulating hepatic glucose production (HGP), which is a major contributor to hyperglycemia in type-2 diabetes (T2D). Results from our study revealed a novel epigenetic mechanism utilized by LncRNA H19 in regulating HGP in both normal and pathological conditions. Functional Study of Long Noncoding RNA H19 in Muscle and Liver Na Zhang B.S., Henan Unversity, 2009 M.S., Institute of Microbiology Chinese Academy of Sciences, 2012 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2018 Copyright by Na Zhang 2018 ii APPROVAL PAGE Doctor of Philosophy Dissertation Functional Study of Long Noncoding RNA H19 in Muscle and Liver Presented by Na Zhang, B.S., M.S. Major Advisor Gordon G. Carmichael Associate Advisor Stormy J. Chamberlain Associate Advisor Yuanhao James Li Associate Advisor Arthur Gunzl University of Connecticut 2018 iii ACKNOWLEDGMENTS My graduate school study experience at University of Connecticut Health Center is an extremely important part of my life. I have had many happy moments, but also experienced a lot of challenges during this period of time. Fortunately, with the help and support from people around me, I was able to overcome those challenges and finish this journey. Therefore, I would like to acknowledge many people who have helped me along the way. First of all, I would like to express my sincere gratitude to my major advisor and mentor, Dr. Gordon Carmichael. He is very open-minded and has encouraged me to work on any project that I was interested in. He guided me to think independently and critically and has always been very supportive. When I want to discuss with him about my project, he would always be ready for a discussion and providing valuable suggestions. Thanks to Gordon’s understanding and generosity, I was able to work on a collaborate project between Gordon’ lab and Dr. Yingqun Huang’s lab at Yale School of Medicine, from which I have gained additional precious research experiences. Other than being a wonderful mentor, Gordon has also been a good friend. There were a lot of happy memories left from our lab parties. I will always be grateful for the guidance, encouragement, support and friendship that Gordon gives to me during my graduate study. I would also like to express my deep gratitude to my external advisor, Dr. Yingqun Huang from Yale school of medicine. Her passion for science and her creative thinking have made it a great pleasure to work with her. She has given me a lot of helpful suggestions on both technique issues and project hypothesis related problems. She has also patiently helped me on improving my presentation skills. I am very grateful to have Dr. Yingqun Huang as my additional advisor while I was working on my thesis project. iv I would like to express my appreciation to my committee members Dr. Stormy J. Chamberlain, Dr. Arthur Günzl and Dr. Yuanhao James Li who have provided many valuable suggestions and constructive advices to me throughout my thesis work. Gordon has attracted a lot of interesting and intelligent people to work with him. I am very glad I have worked in his lab. I would like to express my thanks to the current and past lab members, Kim Morris, Yinzhou Zhu, Ahmad Osman, Stephan Pirnie, Seth Garren, Xiaoxiao Hong, Tulika Sharma, Yuvabharath Kondaveeti and Joseph Autuoro, who have created a lovely working environment in Gordon’s lab. Special thanks to Yinzhou Zhu, who has given me a lot of help on solving my sequencing data analysis related problems. It was also very pleasant to work with the team in Dr. Yingqun Huang’s lab. I would like to express my thanks to Tingting Geng, Ya Liu, Yuanyuan Shen, Zhangsheng Wang, Tiefeng Cao, Ruling Zhang, Liyong Zhu. I would like to thank Tingting Geng and Ya Liu in particular for their significant contributions to our collaborative project. I would also like to thank Gerald I. Shulman and Joao Paulo Camporez from Yale School of Medicine for their help on our hyperinsulinemic/euglycemic clamp studies. I would like to express my special thanks to Dr. Michael Duff and Dr. Justin L. Cotney for their intellectual help and useful discussion on my sequencing data analysis. Finally, I would like to express my special gratitude to my mom, who has always been supportive to me along my growing up, although sometimes at the cost of her own happiness. Without her sustaining encouragement and strong support, I wouldn’t be able to go through many tough times and achieve the goals in my life. I would also like to deeply thank my husband who has been by my side in the last two and half years of my graduate study. Without his company, love, understanding and support, I wouldn’t be able to complete my thesis. v TABLE OF CONTENTS APPROVAL PAGE.........................................................................................................................iii ACKNOWLEDGMENTS...............................................................................................................iv TABLE OF CONTENTS................................................................................................................vi LIST OF FIGURES.........................................................................................................................x LIST OF TABLES..........................................................................................................................xii I. INTRODUCTION A. Long Noncoding RNA...............................................................................................1 B. LncRNA H19...............................................................................................................2 C. Function of LncRNA H19.........................................................................................8 D. H19 Mechanism of Action.....................................................................................10 E. Thesis Objectives....................................................................................................13 II. ROLE OF H19 IN MUSCLE DIFFERENTIATION A. Abstract.....................................................................................................................16 B. Background..............................................................................................................17 C. Material and Methods............................................................................................19 1. Materials.............................................................................................................19 2. Cell culture and transfection..........................................................................19 3. RNA-Seq library construction........................................................................20 4. RNA-Seq data analysis.....................................................................................21 5. RNA extraction and RT-qPCR........................................................................21 6. Western blot analysis......................................................................................21 vi 7. Immunofluorescence analysis......................................................................22 D. Results 1. Knockdown of H19 in differentiating C2C12 cells...................................24 2. RNA-Seq and data analysis............................................................................24 3. RNA-Seq results suggest that H19 negatively regulates muscle differentiation...................................................................................................29 4. H19 knockdown leads to increased expression of muscle terminal differentiation-specific genes........................................................................34 5. H19 knockdown induces precocious myotube formation......................34 E. Discussion.................................................................................................................39 III. ROLE OF H19 IN HEPATIC GLUCOSE PRODUCTION A. Abstract.....................................................................................................................43 B. Background..............................................................................................................44 C. Material and Methods............................................................................................49 1. Materials.............................................................................................................49