CONSTRUCTION OF RECOMBINANT OTUB1 KNOCKDOWN IN BREAST, BONE AND LIVER CANCER CELL LINES by NOR SHILA BINTI MOHAMED NOOR DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE (MEDICAL RESEARCH) ADVANCED MEDICAL AND DENTAL INSTITUTE UNIVERSITI SAINS MALAYSIA 2019 CONSTRUCTION OF RECOMBINANT OTUB1 KNOCKDOWN IN BREAST, BONE AND LIVER CANCER CELL LINES by NOR SHILA BINTI MOHAMED NOOR Dissertation Submitted In Partial Fulfilment Of The Requirement For The Degree Of Master Of Science (Medical Research) ADVANCED MEDICAL AND DENTAL INSTITUTE UNIVERSITI SAINS MALAYSIA 2019 DECLARATION I hereby declare that this research was sent to Universiti Sains Malaysia (USM) for the Degree of Science in Medical Research. It has not been sent to other universities. With that, this research can be used for consultation and photocopied as reference. Sincerely, ……………………………………………. (NOR SHILA BINTI MOHAMED NOOR) P-IPM 0016/18 ii ACKNOWLEDGEMENT First and foremost, I would like to acknowledge and extend my heartfelt gratitude to my supervisor and my co-supervisor, Dr Kumitaa Theva Das and Dr Nurulisa binti Zulkifle for their supervision, guidance, knowledge, motivation and support throughout my dissertation project. Their suggestions and insightful advice are very helpful in spurring me on to complete this dissertation successfully. Besides that, I would like to thank my fellow colleagues in the Infectomics and Oncology Laboratories especially Wahida, Nithya, Auni, Khairi and Asna for their continuous guidance, valuable opinion and knowledge sharing throughout my dissertation project. My deepest gratitude to all staff members in the Infectomics Cluster, Oncology Cluster and Regenerative Cluster laboratories, especially Cik Ira Maya Sofia, Puan Nur Hikmah and Puan Zuliza for their guidance and support in using the laboratory equipment and facilities. Their assistance enables me to run and perform my experiments smoothly. I would like to thank my friend, Athirah and other course mates as well for their encouragement and continuous support until the completion of my dissertation. Last but not least, my sincere thanks to my husband, sons and family members for their understanding and moral support. Their support, unconditional love and encouragement are my driving force that spurs me onwards when I face hurdles during my experimentations. I would also like to express my appreciation to all who had helped me directly or indirectly throughout this study. Without assistance from all mentioned above, I would likely face many difficulties in completing my dissertation. iii TABLE OF CONTENTS DECLARATION……………………………………………………………………ii ACKNOWLEDGEMENT ........................................................................................ iii TABLE OF CONTENTS ..........................................................................................iv LIST OF TABLES .....................................................................................................vi LIST OF FIGURES ................................................................................................. vii LIST OF SYMBOLS ............................................................................................... iix LIST OF ABBREVIATIONS .................................................................................... x ABSTRAK ..................................................................................................................xi ABSTRACT .............................................................................................................. xii CHAPTER 1 INTRODUCTION ........................................................................... 1 1.1 Cancer ............................................................................................................... 1 1.1.1 General Overview of Cancer ............................................................... 1 1.1.2 Breast Cancer ...................................................................................... 2 1.1.3 Bone Cancer ........................................................................................ 3 1.1.4 Liver Cancer ........................................................................................ 4 1.1.5 Epidemiology of Cancer ...................................................................... 5 1.1.6 Cancer Scenario in Malaysia ............................................................... 6 1.2 OTUB1 .............................................................................................................. 8 1.2.1 General Overview ............................................................................... 8 1.2.2 Treatment of Cancer .......................................................................... 11 1.3 Gene Therapy of Cancer and CRISPR ............................................................ 13 1.3.1 Overview of Gene Therapy in Cancer .................................................... 13 1.3.2 CRISPR/Cas9 Systems ...................................................................... 15 1.4 Problem Statement .......................................................................................... 19 1.5 Research Objectives ........................................................................................ 19 iv 1.5.1 Main Objective ................................................................................... 19 1.5.2 Specific Objectives ............................................................................. 19 CHAPTER 2 METHODOLOGY ........................................................................ 20 2.1 Designing and constructing of CRISPR/Cas9 to knockdown OTUB1 ........... 21 2.1.1 Cloning of CRISPR nuclease into expression vector ........................ 21 2.1.2 Verification of CRISPR Sequence .................................................... 24 2.2 Cell Cultures and Reagents ............................................................................. 25 2.2.1 Experimental Design of Transfection Optimization ......................... 26 2.2.2 Protocol of Transfection Optimization Using Lipofectamine 3000 .. 27 2.2.3 Evaluating CRISPR transfection efficiency in cell lines, MCF7, Saos2 and HepG2 using fluorescence microscopy and flow cytometry ........................................................................................... 28 CHAPTER 3 RESULTS ....................................................................................... 30 3.1 Designing and constructing of CRISPR/Cas9 to knockdown OTUB1 ........... 30 3.2 Verification of CRISPR Sequence .................................................................. 32 3.3 Optimization of Transfection Efficiency using Lipofectamine 3000 ............. 34 3.4 Evaluating CRISPR Transfection Efficiency in Cell Lines, MCF7, Saos2 and HepG2 using Fluorescence Microscopy and Flow cytometrey ..... 37 CHAPTER 4 DISCUSSION ................................................................................. 43 CHAPTER 5 CONCLUSION AND FUTURE RECOMMENDATIONS ....... 47 5.1 Conclusion ...................................................................................................... 47 5.2 Recommendations for Future Research .......................................................... 49 REFERENCES .......................................................................................................... 50 v LIST OF TABLES Page Table 2.1 Online Tools Used to Design CRISPR Guide RNA ........................ 20 Table 2.2 Protocol for Transfection Optimization using Lipofectamine 3000..26 vi LIST OF FIGURES Page Figure 1.1 Global Population With Cancer In 2018............................................ 5 Figure 1.2 Number of New Cases In 2018, Both Sexes And All Ages .............. 6 Figure 1.3 Number of New Cases In Males And Females, All Ages In 2018 .... 7 Figure 1.4 A Schematic of The Mechanism of OTUB1 Regulates FOXM ...... 10 Figure 1.5 Schematic of The RNA-Guided Cas9 Nuclease .............................. 16 Figure 1.6 DSB Repair Promotes Gene Editing ................................................ 17 Figure 2.1 Plasmid pSpCas9(BB)-2A-GFP (Px458) ......................................... 21 Figure 2.2 Plasmid Map Of pQBI ..................................................................... 25 Figure 2.3 Experimental Design for Optimization of Transfection Efficiency Using Lipofectamine 3000 ............................................................... 26 Figure 2.4 Experimental Design for CRISPR Transfection In Cell Lines To Test The Efficiency of Transfection Using Lipofectamine 3000 ............ 28 Figure 3.1 Exon Sequence of OTUB1 Gene Sequence ..................................... 31 Figure 3.2 Sequence of gRNA…………………...…………………………….32 Figure 3.3 Gel Electrophoresis Showing That The Positive Colonies All Have The Correct Insert Which Is Approximately 650 Base Pairs ........... 33 Figure 3.4 Chromatogram of The Sequencing Results Which Shows DNA Sequence Contains gRNA ............................................................... 34 Figure 3.5 Result of Optimization of Transfection Using Lipofectamine 3000 With Different Concentrations of DNA and P3000 Reagent For MCF7 ............................................................................................... 35 Figure 3.6 Summary of Result of Transfection Optimization Using Protocol 3 In Different Cell Lines ......................................................................... 36 Figure 3.7 Results of Daily Observation of Transfected MCF7 Cell Lines Under