The Role of Microrna in Cardioprotection: Ischemic Preconditioning and Mesenchymal Stem Cell Paracrine Effects

xviii CHAPTER 1: INTRODUCTION.1 The Role of MicroRNA in Ischemic Preconditioning.1 Myocardial Infarction and Ischemia/Reperfusion Injury.1 Ischemic Preconditioning.4 MicroRNA.10 MiRNA as a Paracrine Mediator from Myocardium to Transplanted Mesenchymal Stem Cells.13 Endogenous Regenerative Potential of the Myocardium . 13 Stem Cell Therapy for Regeneration of the Myocardium . 15 Preconditioning the Heart: a Technique to Enhance Stem Cell Survival? 25 MiRNA in Stem Cell Mediated Paracrine Effects on the Myocardium . 28 Exosome Biogenesis and Transfer . 28 Exosomal Protein Content . 31 Exosomal RNA Content . 32 Summary of Goals and Hypotheses . 38 CHAPTER 2: MATERIALS AND METHODS . 40 Methodology. 40 Animals . 40 Ischemic Preconditioning and Ischemia/Reperfusion Injury . 40 TTC Staining and Infarct Size Assessment. 41 Pericardial Sac Injections . 42 Culture of Cardiac Cell Lines . 42 Culture of Primary Neonatal Mouse Ventricular Myocytes . 43 Culture of Primary Mesenchymal Stem Cells . 45 Hypoxic Preconditioning and Simulated Ischemia Reperfusion Injury.49 Exosome Preparation and Quantification of Uptake. 53 Protein Isolation and Quantification . 54 vi Western Blotting . 56 RNA Isolation . 57 Reverse Transcription . 57 Real-Time PCR . 58 Generation of Luciferase Reporter Plasmids. 59 Transfection and Luciferase Assay . 61 RNA and miRNA-Sequencing . 62 Bioinformatic Analysis of Sequencing Data . 63 In silico Targeting Hypothesis Generation Using Cytoscape . 64 Statistical Analysis . 65 Experimental Limitations . 65 Luciferase Assay Limitations . 65 Limitations of Mimic Transfection and miRNA Knockdown Assays . 66 Imaging-based Viability Assay Limitations . 66 CHAPTER 3: RESULTS . 67 The Role of MicroRNA in Ischemic Preconditioning . 67 Preconditioning and I/R Injury In Vivo and In Vitro . 67 Gene Expression after IPC . 69 Comparison of Gene Expression After IPC and HPC . 75 Identification of miRNAs Acting as Nodes in a Network of Heat Shock Protein Expression . 78 Luciferase Reporter Assays . 82 Knockdown and Mimic Transfection of miRNAs In Vitro . 87 Inhibitor Effects on Viability Upon OGD in HL-1 and H9c2 Cells . 98 Summary of Findings . 100 Exosomal MiRNA as a Paracrine Mediator from Myocardium to Transplanted Mesenchymal Stem Cells . 102 Mesenchymal Stem Cells Internalize HL-1 Exosomes . 102 miRNA-Seq Results . 104 RNA-Seq Results . 106 miRNA in Stem Cell Mediated Paracrine Effects on the Myocardium . 109 MSC Exosomes Are Protective Against Cell Death Upon OGD Challenge . 109 MSC Exosomes Are Internalized by H9c2, HL-1, and NMVM Cells . 111 miRNA Seq Results . 113 mRNA Seq Results . 119 miR-21 Knockout Exosomes Are Less Cardioprotective Than Wildtype Exosomes In Vitro. 120 Reporter Assays for PDCD4 and FasL . 123 PDCD4 3’-UTR Variants . 125 vii Protein and RNA Levels of Predicted miR-21 Target Genes After miR-21 Transfection and Treatment of Cells with Exosomes . 128 Pericardial Sac Injection of WT and KO Exosomes . 131 Expression of miR-21 Target Genes 24h After Exosome Injection and 6h after I/R Injury.132 CHAPTER 4: DISCUSSION . 135 Summary of Hypotheses and Results . 135 Hypothesis 1 . 135 Hypothesis 2 . 136 Hypothesis 3 . 136 The Role of MicroRNA in Ischemic Preconditioning . 137 Gene expression after IPC and HPC . 137 miRNA/HSP Network . 140 Reporter Assays in Support of 3’-UTR Targeting . 142 Knockdown and Mimic Transfection of miRNAs: Effect on HSP Levels. 143 Viability Assays . 145 Conclusions: Interactions of miRNAs and Transcription Factors During Preconditioning . 146 Exosomal miRNA as a Paracrine Mediator from Myocardium to Transplanted Mesenchymal Stem Cells . 149 Hypoxic Preconditioning Regulates HL-1 miRNA . 149 Hypoxic Preconditioning Regulates HL-1 mRNA . 150 Conclusions . 152 miRNA in Stem Cell Mediated Paracrine Effects on the Myocardium . 153 MSC Exosome Mediated Preconditioning . 153 miR-21 Target Genes . 155 Interconnected Regulation of miR-21 and Its Targets . 160 Conclusions . 162 Potential Significance (Basic and Medical) . 163 APPENDIX A: RNA-SEQ DATA, GENES WITH DIFFERENTIAL EXPRESSION BETWEEN SHAM AND IPC MOUSE HEART TISSUE.167 APPENDIX B: RNA-SEQ DATA, GENES WITH DIFFERENTIAL EXPRESSION BETWEEN MESECHYMAL STEM CELLS AND THEIR EXOSOMES . 181 APPENDIX C: RNA-SEQ DATA, HIGHLY EXPRESSED GENES IN MSC EXOSOMES . 204