Title Page
Contents
LIST OF ABBREVIATIONS 8
ABSTRACT 16
Ⅰ. INTRODUCTION 19
Ⅱ. MATERIALS AND METHODS 21
1. Cell lines and cell culture 21
2. Reagents and antibodies 21
3. MITF knockdown in RCC cell lines 22
4. Overexpression of MITF in RCC cell lines 23
5. Transient transfection 23
6. In vitro cell proliferation assay 24
7. Anchorage-independent growth assay (Anoikis assay) 24
8. Clonogenic assya 24
9. Migration and invasion assays 25
10. Western blot analysis 26
11. Wound healing assay 26
12. Immunofluorescence 27
13. Cell cycle analysis 28
14. Active RhoA/Rac1/Cdc42 pulldown assay 28
15. Xenograft tumors in nude mice 29
16. Statistical analysis 29
Ⅲ. RESULTS 31
1. Knockdown of MITF inhibits cell growth by cell cycle shift. 31
2. Knockdown of MITF suppresses cell migration and invasion in vitro. 40
3. Knockdown of MITF inhibits YAP nuclear translocation. 46
4. Knockdown of MITF suppresses RhoA activation. 56
5. MITF overexpression promoted cell migration and invasion by upregulating RhoA/YAP signaling. 61
6. MITF affects tumorigenesis in ccRCC cells in vivo. 70
Ⅳ. DISCUSSION 75
Ⅴ. REFERENCES 79
국문초록 87
Figure 1. Expression of MITF in clear cell renal cell carcinoma cell lines (ccRCC). 33
Figure 2. MITF-knockdown in ccRCC. 34
Figure 3. MITF-knockdown inhibits cell proliferation. 35
Figure 4. MITF-knockdown inhibits anchorage-independent growth and colony formation. 36
Figure 5. MITF-knockdown inhibits cell growth via a cell cycle shift in the ccRCC. 39
Figure 6. MITF-knockdown impairs cell motility in a wound healing assay. 42
Figure 7. MITF-knockdown reduces phalloidin expression. 43
Figure 8. MITF-knockdown reduces migration and invasion capability. 45
Figure 9. The relationship of MITF-knockdown and YAP expression in ccRCC. 48
Figure 10. MITF-knockdown regulates YAP expression. 49
Figure 11. YAP overexpression in MITF-knockdown cells (I). 50
Figure 12. YAP overexpression in MITF-knockdown cells (II). 52
Figure 13. YAP overexpression increases cell cycle related protein in MITF-knockdown cells. 53
Figure 14. YAP overexpression increases cell migration in MITF-knockdown cells. 54
Figure 15. YAP overexpression increases cell invasion in MITF-knockdown cells. 55
Figure 16. Knockdown of MITF suppresses GTPase family activation. 57
Figure 17. RhoA activation increases the expression of phospho-FAK and YAP. 58
Figure 18. RhoA activation increases cell cycle related protein in MITF-knockdown cells. 59
Figure 19. Knockdown of MITF suppresses GTPase family activation. 60
Figure 20. Positive correlation between MITF and RhoA, CRIK, CDK2, and YAP1 expression in the GEPIA kidney ccRCC dataset. 63
Figure 21. MITF overexpression increases FAK phosphorylation, and expression of CRIK, RhoA and YAP 64
Figure 22. MITF overexpression increases cell cycle related protein. 65
Figure 23. MITF overexpression increases cell migration and invasion. 66
Figure 24. RhoA/YAP signaling is dependent MITF. 67
Figure 25. RhoA or YAP-knockdown decreases cell migration in MITF-overexpression cells. 68
Figure 26. RhoA or YAP-knockdown decreases cell invasion in MITF-overexpression cells. 69
Figure 27. MITF-knockdown affects tumorigenesis in ccRCC in vivo (I). 71
Figure 28. MITF-knockdown affects tumorigenesis in ccRCC in vivo (II). 72
Figure 29. Overexpression MITF affects tumorigenesis in ccRCC in vivo. 73
Figure 30. Representative images of H&E, Ki-67 and MITF staining. 74