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Mirnas As Biomarkers for Prostate Cancer Progression Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2015 MIRNAS AS BIOMARKERS FOR PROSTATE CANCER PROGRESSION Gene C. Clark Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Diagnosis Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/3954 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. MIRNAS AS BIOMARKERS FOR PROSTATE CANCER PROGRESSION A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Virginia Commonwealth University By Gene Chatman Clark B. S. 2012, Virginia Polytechnic Institute Director: Zendra E. Zehner Professor, Department of Biochemistry Virginia Commonwealth University Richmond, Virginia July 22, 2015 Acknowledgement Firstly, I would like to thank the many people here at VCU have inexplicably taken a chance on me. This includes, but could not possibly be limited to, Dr. Kimberly Jefferson who let me work in her lab when I was just a non-degree seeking student, Dr. Shirley Taylor who successfully encouraged me to pursue my love of research, and Dr. Louis DeFelice who suggested that I apply to the Pre-Medical Certificate Program here at VCU. I would also like to thank my amazing lab mates, Qianni Wu and Rhonda Daniel, whose insight and support were invaluable to this project. I have been lucky to have had them as partners and as friends. I would also like to thank Dr. Mike Holmes for his advice and for all of the time that he spent goofing off with me in the break room. Finally, I would like to thank Dr. Zendra Zehner for her much needed guidance. I could not have gotten anywhere without her. Because she is modest, she will tell you a different story. Do not believe her. ii Table of Contents List of Figures ……………………………………………………………………………………vi List of Tables ……………………………………………………………………………………vii Abbreviations and Symbols ……………………………………………………………………...ix Abstract …………………………………………………………………………………………...1 Introduction ……………………………………………………………………………………….3 The Prostate ………………………………………………………………………………3 Prostate Cancer …………………………………………………………………………...3 The Relevance of miRNAs ……………………………………………………………….4 Extracellular miRNAs and the Tumor Microenvironment ……………………………….4 Canonical and Non-Canonical Pathways …………………………………………………5 miRNA Action ……………………………………………………………………………7 miRNA Expression and Regulation ………………………………………………………8 3p vs 5p ………………………………………………………………………………….10 The General Silencing of miRNAs in Cancer …………………………………………...11 The Epithelial-Mesenchymal Transition ………………………………………………...13 Why Next Generation Sequencing (NGS) ………………………………………………13 Chapter 1: A Urinary Assay for the Detection of Prostate Cancer ……………………………...15 Specific Aims ……………………………………………………………………………16 Urine Over Blood and Semen …………………………………………………………...17 Materials and Methods ………………………………………………………………..…18 RNA Extraction from Urine ……………………………………………………..18 Illumina ……………………………………………………………………….…20 RT- PCR …………………………………………………………………….......21 Results …………………………………………………………………………………...22 NGS Normalization ……………………………………………………………..22 iii RNA-sequencing ………………………………………………………...………27 Possible prostate specific miRNAs: 126-3p …………………….………………30 Normalization of qRT PCR Analysis of Urine ………………….………………31 Why Normfinder ………………………………….……………………..………34 qPCR samples …………………………………………………..…….…………36 Accuracy vs Area Under the Curve ………………………..……………………38 qRT-PCR Results …………………………………………………..……………40 Discussion ……………………………………………………………….………………60 The Assay ……………………………………………….……………….………60 Contemporary Attempts at a Urinary Test for pCa …………...…………………61 The Urinary Panel ………………………………………………….……………62 Hsa-miR-204-5p ………………………………...………………………62 Hsa-miR-205-5p …………………………………………...……………63 Hsa-miR-223-3p and hsa-miR-223-5p ……………….…………………64 Hsa-miR-199a-3p ………………………………………….….…………65 Hsa-miR-126-3p …………………………………………...……………66 Hsa-miR-143-3p ……………………………………………………...…66 Hsa-miR-184-5p …………………………………………………...……67 The Question of Normalization for a Clinical Assay ……………………………67 How miRNAs might get into the Urine and the Potential for Outliers …….……68 Thoughts for the Future …………………………………………………………70 Indolent vs. Aggressive forms of Disease ………….……………………70 Metastasis …………………………………………….………….………71 The Androgen receptor and CRPC ……………………...………………72 The Big Data Problem ……………………………………..……………73 Prostate Cancer Subgroups …………………………………….…..……74 Tissue Specific miRNA Modifications …………………………….……74 Detecting the expansion of specific miRNA profiles ……………...……………75 iv Delivery to the Vagina …………………………………………..………………76 Chapter 2: miRNA NGS of the Cancer Cell Line Progression Model …………………….……78 Specific Aims ……………………………………………………………………………79 History and Character of the Cell Lines …………………………………………...……79 Materials and Methods ………………………………………………………………..…82 Cell Line RNA Extraction and Sequencing …………………………………..…82 TCGA pCa tumor samples ………………………………………………………83 Results ………………………………………………...…………………………………84 SNP Analysis ……………………………………………………………………84 RNA Sequencing …………………………………………………………..……89 Discussion ……………………………………………………………………………...108 SNP Analysis …………………………………………………………..………108 The Early Downregulated miRs ……………………………………………..…109 The Chromosome 14 Cluster ………………………………………..…109 The miR-200 Family …………………………………………………...112 The Late Upregulated miRNAs ……………………………………………..…119 miR10a and miR-196a …………………………………………………119 miR-675 and lncRNA-H19 ……………………………………….……122 The 548 Family ………………………………………………………...124 miR-34a-5p ……………………………………………………….……125 The Rest ………………………………………………………..………………128 miR-9 ………………………………………………………………..…128 miR146a-5p …………………………………………….………………130 Literature Search- Tumor suppressors lost with chromosome 19p ……………131 Low vs. High copy numbers ………………………...…………………………135 Oncomirs and Tumor Suppressors miRs: The Reductionist’s Approach …...…136 Thoughts for the Future- Considering Proteomic Data ………………………...138 Bibliography …………………………………………………………………...………………141 Appendix …………………………………….…………………………………………………152 Vita ……………………………………………………………………………………………..175 v List of Figures Figure 1: A comparison between samples of RNA in small size range ………………………..25 Figure 2: RT-PCR Analysis for miR-204-5p ………………………………….………………..41 Figure 3: RT-PCR Analysis for miR-205-5p ………………………………………….………..43 Figure 4: RT-PCR Analysis for miR-223-3p …………………………………………………...45 Figure 5: RT-PCR Analysis for miR-199a-3p ………………………………………………….48 Figure 6: RT-PCR Analysis for miR-223-5p …………………………………………………...50 Figure 7: RT-PCR Analysis for miR-126-3p …………………………………………………...52 Figure 8: RT-PCR Analysis for miR-143-3p …………………………………………………...54 Figure 9: RT-PCR Analysis for miR-184-5p …………………………………………………...56 Figure 10: The combined assay ………………………………………………………………...59 Figure 11: The chromosomal representation of dysregulated miRNAs between the P69 and M2182 cell lines …………………………………………………………………………………94 Figure 12: The chromosomal representation of dysregulated miRNAs between the P69 and M12 cell lines ………………………………………………………………………………………..101 Figure 13: The chromosomal representation of dysregulated miRNAs between the P69 and F6 cell lines ………………………………………………………………………………………..106 Figure 14: Suppression of the Dlk-Dio3 megacluster in prostate cancer ………………….….110 Figure 15: The Expression of the miR-200 Family and associated miRNAs over the Cancer Progression Model ……………………………………………………………………………..114 Figure 16: The Expression of miR-10a and miR196a (3p and 5p) in the Cancer Cell Line Progression Model ……………………………………………………………………………..119 Figure 17: The Expression of miR-675 in the Cancer Cell Line Progression Model ..………..122 Figure 18: The Expression of miR-34a in the Cancer Cell Line Progression Model ..………..125 Figure 19: The Expression of miR-9 in the Cancer Cell Line Progression Model ……………128 Figure 20: The Expression of miR-146a in the Cancer Cell Line Progression Model ………..130 vi List of Tables Table 1: Candidates for RNA Sequencing ………………………….………………………..27 Table 2: Sequencing Statistics ………………………………………………………………...28 Table 3: microRNA Sequencing Results of Urine Samples …………………………………29 Table 4: NGS Sequencing Results for Normalization Candidates ………………………….33 Table 5: Normfinder Stability Values ………………………………………………………...34 Table 6: Samples Used for the First Round of Validation …………………………………..37 Table 7: Additional Samples used for the Second Round of Validation …………………....38 Table 8: Summary of qRT-PCR Results ………………………….……………………….…40 Table 9: Final Assay Statistics ………………………………………………………………...59 Table 10: SNP NGS Summary ………………………….……………………………………..84 Table 11: SNP Summary ……………………………………………………………………....85 Table 12: SNP Allelic Frequencies in the P69 Cell Line ……………………………………..86 Table 13: SNP Allelic Frequencies in the M2182 Cell Line …………………………………87 Table 14: SNP Allelic Frequencies in the M12 Cell Line ……………………………………88 Table 15: SNP Allelic Frequencies in the F6 Cell Line ……………………………………....89 Table 16: miRNA NGS P69 vs M2182 ………………………………………………………..90 Table 17: Sequencing Results M12 ……………………………………………………………95 Table 18: P69 vs F6 …………………………………………………………………………...102 Table 19: Overall Sequencing Summary of miR expression in the parental p69 Cells compared to its subsequent variants ……………………………………………………..….107 Table 20: RPMA Analysis Shows Increased Protein Expression from the PI3K/RAS pathways in M12 versus p69 Cell Lines Table 21: Extended
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