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RNA Sequencing in the Development of Cancer-Cachexia" (2017)

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RNA Sequencing in the Development of Cancer-Cachexia University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2017 RNA Sequencing in the Development of Cancer- Cachexia Thomas Allen Blackwell University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Cancer Biology Commons, Community Health and Preventive Medicine Commons, and the Kinesiology Commons Recommended Citation Blackwell, Thomas Allen, "RNA Sequencing in the Development of Cancer-Cachexia" (2017). Theses and Dissertations. 2434. http://scholarworks.uark.edu/etd/2434 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. RNA Sequencing in the Development of Cancer-Cachexia A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Kinesiology by Thomas Blackwell Oklahoma State University Bachelor of Science in Health Education and Promotion, 2015 August 2017 University of Arkansas This thesis is approved for recommendation to the Graduate Council. ____________________________ Dr. Nicholas Greene Thesis Director ____________________________ ____________________________ Dr. Tyrone Washington Dr. Walter Bottje Committee Member Committee Member Abstract Introduction: Cancer is a major public health problem in the U.S. and the world. In 2013 there were an estimated 1,660,290 new cases of cancer in the U.S. Cancer-Cachexia (CC) is a common effect of many cancers, and is directly responsible for 20-40% of cancer-related deaths. The mechanisms that control the development of CC are not well understood. Most investigations of CC focus on the post-cachectic state and do not examine the progression of the condition. The purpose of this study was to utilize RNA sequencing to analyze transcriptomic alterations throughout the progression of CC. Methods: Lewis Lung Carcinoma cells (LLC) or Phosphate Buffered Saline (PBS, control) were injected into the hind-flank of wildtype C57BL6/J mice at 8 wks of age, and tumor allowed to develop for 1, 2, 3, or 4 wks before euthanasia. RNA was isolated from the gastrocnemius and RNA sequencing performed. Results: RNA sequencing showed widespread alterations in LLC when compared to PBS animals with largest differences seen in 4 wk LLC compared to other conditions. Commonly altered pathways included: Oxidative Phosphorylation, Mitochondrial Dysfunction, and Protein Ubiquitination. Discussion: We demonstrated that alterations in the muscle belly likely occur in phases beginning with early mitochondrial degenerations, which now appear to lead to large transcriptomic shifts concurrent to the onset of muscle atrophy. In agreement with previous work, we observed multiple aspects related to degeneration of mitochondria and oxidative metabolism. The early onset of these alterations shows a need for early interventions in order to effectively ameliorate the effects of CC. Formatted for submission in FASEB J. Acknowledgements I would like to thank collaborators from Integrative Muscle Metabolism Laboratory: my advisor Dr. Nicholas Greene, Jacob Brown, Megan Rosa, and David Lee. I would also like to thank collaborators from the Exercise Muscle Biology Laboratory: Dr. Tyrone Washington, Dr. Lemuel Brown, Wesley Haynie, and Richard Perry, Jr. I would also like to thank collaborators from the Department of Poultry Science: Dr. Bhuwan Khatri and Dr. Byung-Whi Kong. I would also like to thank Dr. Michael Wiggs from the Integrated Physiology and Nutrition Laboratory at the University of Texas at Tyler. I would also like to thank Dr. Igor Cervenka and Dr. Jorge Ruas from Molecular and Cellular Exercise Physiology department of Karolinska Institutet. I would like to thank Mr. Kevin B. Greene for his assistance with this project. Furthermore, we would like to extend our gratitude to the numerous faculty, staff, and students of the Exercise Science Research Center at the University of Arkansas. Without your help this project would not have been possible. Dedication I would like to dedicate this thesis to my wife, Mrs. Meredith Blackwell. Thank you for being an endless source of support and encouragement. Table of Contents Cancer-Cachexia Overview .......................................................................................................... 1 Known Pathways of Muscular Atrophy in Cancer-Cachexia ............................................... 2 Protein Synthetic Pathways ..................................................................................................... 3 Myogenic Pathways ................................................................................................................ 3 Protein Degradation ................................................................................................................ 4 Inflammatory Pathways .......................................................................................................... 6 Mitochondrial Modulation ...................................................................................................... 7 Current Pharmacological Interventions in Cancer-Cachexia .............................................. 8 Summary of Known Mechanisms and Treatments for Cancer-Cachexia ......................... 10 Purpose ..................................................................................................................................... 11 Objectives for Data Analysis .................................................................................................. 11 Materials and Methods ............................................................................................................... 13 Animals and Interventions ..................................................................................................... 13 RNA Isolation and Quality ..................................................................................................... 13 RNA Sequencing and Data Analysis ..................................................................................... 14 Comparison of RNA Sequencing to the MitoCarta ............................................................. 15 Statistical Analysis .................................................................................................................. 15 Results .......................................................................................................................................... 16 Phenotypic description of LLC-induced muscle atrophy across time course ................... 16 RNA Sequencing effectively discriminates cachectic vs. non-cachectic animals .............. 16 Differentially expressed genes appeared primarily in comparisons to Week 4 LLC mice ................................................................................................................................................... 17 Pathway analysis of differentially expressed genes for all comparisons ............................ 17 Analysis of transcription factors common to DE genes among comparisons .................... 18 Discussion ..................................................................................................................................... 19 Summary .................................................................................................................................. 22 Objectives for Data Analysis .................................................................................................. 23 References .................................................................................................................................... 24 Figures and Tables ...................................................................................................................... 31 Legends: ................................................................................................................................... 31 Table 1: .................................................................................................................................... 32 Figure 1: ................................................................................................................................... 32 Figure 2: ................................................................................................................................... 33 Table 2: .................................................................................................................................... 33 Figure 3: ................................................................................................................................... 34 Supplemental Figure 1: .......................................................................................................... 35 Figure 4: ................................................................................................................................... 36 Figure 5: ................................................................................................................................... 37 Table 3: .................................................................................................................................... 37 Figure 6: ................................................................................................................................... 38 Appendix .....................................................................................................................................
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