HIGH-THROUGHPUT SCREENING IDENTIFIES MICRORNAS THAT TARGET NOX2 AND IMPROVE FUNCTION FOLLOWING ACUTE MYOCARDIAL INFARCTION Open Access

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that can regulate gene expression by inducing either degradation or translational inhibition of a target mRNA. miRNAs have been indicated to regulate up to 90% of human genes and played significant role in heart diseases.

Myocardial infarction (MI) is the most common cause of heart failure. Excessive production of reactive oxygen species (ROS) plays a key role in the pathogenesis of cardiac remodeling after MI. NADPH with Nox2 as the catalytic subunit is a major source for cardiac superoxide production. Nox2 expression is significantly increased in the infarcted area, especially in macrophages and myocytes. Mice lacking the Nox2 gene are protected from heart injury.

Here we demonstrate to utilize the screening of the miRNA-targets using self-assembled cell (SAMcell) microarray to identify miRNAs that could regulate Nox2 expression and select three miRNAs, miR-106b, miR-148b, and miR-204, for further study. We use different in vitro assays to validate these miRNAs function on Nox2 expression and downstream products. After that we use acid-degradable polyketal particles that could effectively encapsulate miRNAs and deliver them into macrophages. Both in vitro and in vivo studies confirmed the PK3-miRNAs particles could inhibit Nox2 expression and activity, and significantly improve cardiac function. These results revealed new miRNAs for heart disease treatment and provided an effective strategy from miRNAs identify to in vivo delivery.

Our study successfully combine the miRNAs high throughput screening system and macrophages specific delivery system and establish an easy and efficient method from screening to drug delivery. We validate this system and the selected miRNAs functions both in vitro and in vivo. This provides us new evidence and directions for not only myocardial infarction recovery, but also other inflammation related diseases.

Table of Contents

TABLE OF CONTENTS

ACKNOWLEDGEMENTS iii

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF SYMBOLS AND ABBREVIATIONS ix

SUMMARY xii

CHAPTER 1. INTRODUCTION 1

1.1 The Significance of Cardiovascular Disease and Research: 1

1.2 Ischemic Injury After Myocardial Infarction 2

1.3 Inflammation 5

1.4 Cardiac Remodelling 7

1.5 ROS and Nox2 in MI 12

1.6 miRNA 13

1.7 High-throughput miRNA-targets screening system 17

1.8 Polyketal PK3 Nanoparticles. 19

CHAPTER 2. SAMCell Screening To Target Human Nox2 23

2.1 Fabrication of SAMCell 23

2.2 Construction of Report System 24

2.3 miRNA-targets screening 24

2.4 Screening results 25

CHAPTER 3. Selected miRNAs Functional Validation 29

3.1 Cell Culture 29

3.2 miRNA Transfection 29

3.3 Luciferase Assay 31

3.4 RNA Isolation 32

3.5 Complementary DNA (cDNA) Synthesized 34

3.6 Real time PCR 36

3.7 Intracellular Protein Isolation. 37

3.8 Western Blot 38

3.9 Results 40

CHAPTER 4. In Vitro Functional Knockdown of Nox2 Downstream Production 43

4.1 Superoxide Production Staining with Probe 43

4.2 Results 43

CHPATER 5. Nanoparticle Uptake by Macrophages 46

5.1 Polyketal (PK3) Synthesis 46

5.2 Preparation of miRNA-loaded PK3 particles 47

5.3 In vitro delivery of PK3-miRNA particles 48

5.4 Results 48

CHPATER 6. PK3-miRNAs Nanoparticles In Vivo Delivery 51

6.1 Myocardial Infarction and Particle Injection 51

6.2 Immunohistochemistry 52

6.3 Echocardiography 52

6.4 Results. 53

CHPATER 7. Discussion 58

CHPATER 8. Future Work 70

CHPATER 9. Other Project During Ph.D. Program: Biocompatibility Assessment of Detonation Nanodiamond in Non-Human Primates Using Urine, Hematologic, and Histological Analysis 71

9.1 Summary 71

9.2 Introduction 72

9.2.2 Synthesis of Nanodiamonds 73

9.2.3 Properties of Nanodiamonds 74

9.2.4 Applications of Nanodiamonds 76

9.3 Methods for Detonation Nanodiamond Administration in A Non-human Primate Model 78

9.3.1 Non-human Primate Care and Identification 78

9.3.2 Non-human Primate Dosing Protocol 79

9.3.3 Non-Human Primate Serum and Urine Analysis 80

9.3.4 Non-Human Primate Histopathology Analysis 80

9.3.5 Statistics 81

9.4 Results 81

9.4.1 Body Weight Analysis after DNDs Administration 81

9.4.2 Complete Blood Count Analysis after DNDs Administration 82

9.4.3 Urinalysis Following DND Administration 86

9.4.4 Non-human Primate Histopathology Analysis 89

9.5 Conclusion 90

9.6 Discussion 91

Appendix 95

References 108

About this Dissertation

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School	Laney Graduate School
Department	Biomedical Engineering
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Engineering, Biomedical
Keyword	Myocardial infarction Nox2 microRNA Macrophage ROS
Committee Chair / Thesis Advisor	Davis, Michael E, Emory University Xi, Jianzhong, BME, Peking University
Committee Members	Haifeng, Chen, BME, Peking University Junbiao, Dai, BS, Tsinghua University Yangming, Wang, BS, Peking University Li, Changhui, BME, Peking University

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