Title Page
Abstract
Contents
List of abbreviations 11
Introduction 16
Breast cancer 16
Treatment of breast cancer 16
Limitation of TNBC treatment 17
RNA-binding proteins 18
Implications of RBPs in cancer 19
RBPs as therapeutic targets in cancer 20
Purpose of the study 22
Materials and Methods 23
Cell lines 23
Cell proliferation assay 23
Colony forming assay 23
Cell migration and invasion 23
Wound healing assay 23
Flow cytometry cell cycle analysis 24
Sphere formation assay 24
Immunoprecipitation and western blotting 24
Immunofluorescence assay 24
Short hairpin RNA 25
Plasmids and luciferase assay 25
Microarray analysis 26
Quantitative real time polymerase chain reaction 26
Data processing 26
Statistical analysis 27
Hierarchical clustering 27
Chromatin immunoprecipitation assay 27
Short interfering RNA 28
RNA-immunoprecipitation 28
Preparation of the chitosan-nanoparticle 28
Antitumor effectiveness of CH-NP-NONO siRNA. 29
Protein structural homology modeling 29
High throughput drug screening 29
Chemicals 30
Caspase 3/7 activity assay 30
Tissue microarray and immunohistochemistry 30
Results 31
Identification of NONO as an oncogenic RBP in TNBC 31
NONO influences TNBC cell growth 40
NONO regulates STAT3 expression in TNBC 49
Mechanism of STAT3 regulation by NONO in TNBC 56
Clinical significance of the NONO-STAT3 interaction in TNBC 69
Silencing of NONO enhances the sensitivity of TNBC cells to chemotherapy 76
Drug screening for inhibitors targeting NONO in TNBC 87
Discussion 94
Identification of the molecular function of NONO in TNBC 94
NONO as therapeutic targets for TNBC 96
Conclusions 100
References 101
국문요약 112
Figure 1. NONO as TNBC-specific RBP. 34
Figure 2. NONO is associated with drug response in TNBC. 36
Figure 3. NONO expression in TNBC. 37
Figure 4. NONO expression is linked to patient survival in TNBC. 39
Figure 5. NONO regulates cell growth in TNBC cells. 42
Figure 6. NONO modulates cell cycle and caspase3/7 activity in TNBC cells. 44
Figure 7. Depletions of NONO and its effect on migration. 46
Figure 8. Depletions of NONO and its effect on invasion. 47
Figure 9. Depletions of NONO and its effect on BC tumorigenesis. 48
Figure 10. NONO regulates the cellular growth and movement associated genes in TNBC. 51
Figure 11. NONO regulates STAT3 expression in TNBC. 53
Figure 12. NONO regulates STAT3 gene expression and thereby governs TNBC cell growth. 55
Figure 13. NONO binding sites in the STAT3 RNA. 59
Figure 14. NONO binding sites affect STAT3 luciferase activity. 61
Figure 15. NONO physically interacts with STAT3. 62
Figure 16. NONO colocalizes with STAT3 in TNBC cells. 64
Figure 17. The direct interaction between NONO and STAT3 enables STAT3 to function as a transcription factor. 66
Figure 18. NONO directly mediates the function of STAT3 in TNBC cells. 68
Figure 19. STAT3 expression was linked to a bad prognosis and correlated with NONO expression in TNBC. 71
Figure 20. The clinical association between NONO and STAT3 in TNBC. 72
Figure 21. NONO-STAT3 regulates the cellular growth and movement associated gene in TNBC. 73
Figure 22. Patient prognosis was predicted based on the expression of NONO-STAT3 using a prediction model. 75
Figure 23. Survival rate analysis of BC patients according to NONO expression levels. 79
Figure 24. Clinical relevance of NONO and STAT3 in BC. 80
Figure 25. Correlation of NONO and cancer stem cell (CSC) marker in BC. 82
Figure 26. Inhibition of NONO expression enhances the responsiveness of TNBC cells to chemotherapy and radiation. 84
Figure 27. NONO induces drug resistance in TNBC cells through the regulation of the STAT3 gene. 86
Figure 28. FDA-approved drug screening for NONO inhibitors in TNBC. 89
Figure 29. NONO inhibitory effect of Auranofin in TNBC. 91
Figure 30. Modeling of the structure prediction of drug-target interaction. 93