声明
List of Abbreviations
Chapter One Introduction
1.1 Ubiquitination and UBLs
1.2 NEDD8 and neddylation
1.3 Effects of NEDD8 conjugation on its substrates
1.4 Changes of proteins level in cancer cells upon neddylation inhibition
1.5. Neddylation inhibition modulates the tumor microenvironment and impedes tumor growth
1.6 Effects of neddylation inhibition on different types of cancers
1.6.1 Blood cancer
1.6.2 Liver Cancer
1.6.3 Brain Tumor
1.6.4 Breast Cancer
1.6.5 Colon cancer
1.6.6 Esophagus cancer
1.6.7 Gastric cancer
1.6.8 Hyperplasia
1.6.9 Lung Cancer
1.6.10 Melanoma
1.6.11 Osteosarcoma
1.6.12 Prostate cancer
1.6.13 Renal Carcinoma
1.7 UBE2M and importance of NAE-UBE2M interaction inhibition for cancer treatment
1.8 Available Neddylation inhibitor
1.9.1 Objective
1.9.2 Methods follow for drug repurposing
1.9.3 Identified repurposed drugs for cancer treatment
1.9.4 Identified repurposed drugs as ubiquitin ligase inhibitors
1.10 Micafungin Sodium
1.11 Otilonium Bromide
Chapter Two Establishment of HTRF assay for high-throughput drug screening
2.1 Constructions of the recombinant plasmid with NAE, UBE2M andNEDD8 gene
2.1.1 Objective
2.1.2 Plasmid construction
2.2 Protein expression and purification
2.2.1 Materials and reagents
2.2.2 Experimental procedure
2.2.3 Results and Discussions
2.3 Established HTRF based drug screening method and perform high-throughput drug screening
2.3.1 Assay principal
2.3.2 Materials and reagents
2.3.3 Experimental procedure
2.3.4 HTRF data analysis
2.3.5 Results and Discussions
2.4 Summary
Chapter Three Evaluate the effects of Micafungin Sodium and Otilonium Bromide on gastric cancer cells
3.1 Objective
3.2.1 Reagents
3.2.2 Instruments
3.2.3 Preparation of working solutions
3.3 Cell lines
3.4 Experimental procedure
3.4.1 In-vitro neddylation with compound
3.4.2 Immunoprecipitation
3.4.3 Cell culture
3.4.4 Cell lysis and protein extraction
3.4.5 BCA Assay
3.4.6 Colony formation assay
3.4.7 Wound healing assay
3.4.8 Immunofluorescence assay
3.4.9 Cell cycle Analysis
3.4.10 Trans-well Migration assay
3.4.11 MTT assay
3.4.12 Cell viability assay
3.5 Results and Discussions
3.5.1 Micafungin sodium interact with UBE2M and Otilonium bromide with NAE
3.5.2 Gastric cancer cell screening
3.5.3 Micafungin S and Otilonium B inhibited UBE2M-NEDD8 interaction and global cullin neddylation in the cellular level
3.5.4 Micafungin S and Otilonium B decreased the viable cell number and inhibited the colony formation capacity of cancer cells
3.5.5 Micafungin S and Otilonium B have the ability to inhbiti cell migration
3.5.6 Micafungin S and Otilonium B impeded epithelial-mesenchymal transition (EMT) by upregulating E-cadherin and down-regulating Snail
3.5.7 Micafungin S and Otilonium B induced DNA damage and cell cycle arrest in G2/M checkpoin
3.6 Summary
Conclusions
参考文献
Biography
Research papers publication during masters studies
致谢
郑州大学;
