The study for the oncogenic effect of NGEF
- Issued Date
- 2007
- Abstract
- The activated Ras oncogene can transform various mammalian cells and has been implicated in development of a high population of malignant human tumors. The mechanism by which Ras induces tumor progression is, however, not fully elucidated. In this report, we found that the levels of neuronal guanine nucleotide exchange factor (NGEF) mRNA and protein are significantly increased in oncogenic H-RasV12-transformed NIH3T3 cells. The levels of NGEF mRNA and protein were decreased in H-RasV12-transformed cells transient transfected with a dominant negative form H-RasN17, and treatment of ERK and PI3K inhibitors led to significant suppression of oncogenic H-Ras-induced NGEF expression. In addition, the expression of NGEF were capable of activating the small GTPase (Rho, Rac1, Cdc42), and transfection of NGEF siRNA into H-RasV12-transformed cells resulted in a decrease in the activity of small GTPase (Rho, Rac1, Cdc42).
To investigate the biological function of oncogenic H-Ras-induced NGEF expression, we examined whether NGEF is involved in oncogenic H-Ras-mediated increase in cancer progression. We found that the abilities of cellular proliferation, colony formation in soft agar and aggregation of NGEF expressing cells were significantly increased as compared with those of empty vector transfected cells. The abilities of cellular proliferation, colony formation and aggregation of H-RasV12-transformed NIH3T3 cells were significantly suppressed by transfection of NGEF siRNA. We further demonstrated that the transfection of NGEF siRNA into H-RasV12-transformed NIH3T3 cells led to suppression of in vitro cellualr migration, invasion and angiogenesis. In addition, NGEF siRNA transfected H-RasV12-transformed cells exhibited significant reduction of animal tumor growth, angiogenesis and metastasis, wherase control siRNA transfectants did not. Moreover, silencing of NGEF in H-RasV12-transformed cells led to increase of animal survival rate. These results suggest that NGEF is a novel downstream target protein of oncogenic H-Ras, and oncogenic H-Ras-induce NGEF expression may be important role for oncogenic H-Ras-mediated tumor progression.
- Alternative Author(s)
- Kim Hong Beum
- Department
- 일반대학원 생물신소재학과
- Awarded Date
- 2008-02
- Table Of Contents
- CONTENTS
Contents.......................................................................ⅰ
List of Figures...............................................................ⅴ
List of Abbreviations................................................................ⅶ
ABSTRACT....................................................................1
I. INTRODUCTION
1. Ras proteins function as membrane-associated GTPase switches........................................................................3
2. Ras functions as a signaling node............................................................................12
3. Ras deregulation of cell proliferation...................................................................24
4. Ras targets involved in tumor cell Angiogenesis, Invasion and Metastasis.............................................................27
5. Rho GTPases in tumorigenesis...................................35
II. MATERIALS AND METHODS
1. Reagents and Antibodies................................39
2. Cell culture...................................................39
3. Plasmid constructs and Transfection...............40
4. Small interfering RNA (siRNA)-based experiments..................................................................40
5. Semiquantative reverse transcriptase?Cpolymerase chain reaction ∼.......................................42
6. Western blotting.............................................43
7. DEG (Differentially Expressed Gene) experiment...................................................................44
8. Small GTPase activation assay......................45
9. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay......................46
10. Serum stimulation and BrdU labeling...............46
11. Cellular aggregation assay............................47
12. Soft agar colony formation analysis................48
13. In vitro Migration assay using transwell...........48
14. Wound healing migration assay......................49
15. In vitro Invasion assay...................................49
16. In vitro Angiogenesis assay............................50
17. Tumorigenesis in nude mice..........................50
18. Immunofluorescent and immunohistochemical staining........................................................................51
19. Laser scanning microscopy and image analysis.......................................................................52
20. Animal micrometastasis assay.......................53
21. Animal survival assay....................................53
22. Statistical analysis.........................................54
III. RESULTS
1. Identification of differential gene expression in NIH3T3 cells and oncogenic H-RasV12-transformed NIH3T3 cells............................................................................55
2. Oncogenic H-Ras induces expression of the NGEF...........................................................................58
3. Dominant negative H-RasN17 suppressed H-RasV12-induced NGEF expression..................................60
4. Effect of Ras signal pathways inhibition on NGEF expression in oncogenic H-RasV12-transformed cells.......62
5. Effect of NGEF on the Raf, Rho, Rac1 and Cdc42 activation......................................................................64
6. EGF-stimulated induction of NGEF is dependent upon ERK, PI3K.............................................................67
7. NGEF is required for oncogenic H-Ras-induced cellular proliferation........................................................69
8. Effect of NGEF on the Cell cycle regulator proteins........................................................................71
9. Effect of NGEF on the Aggregation in NIH3T3 and oncogenic H-RasV12-transforming NIH3T3 cells................74
10. Effect of NGEF on the Colony formation in NIH3T3 and oncogenic H-RasV12-transforming NIH3T3 cells ...........................................................................76
11. Effect of NGEF on the Migration in NIH3T3 and oncogenic H-RasV12-transforming NIH3T3 cells...............79
12. Effect of NGEF on the Invasion in NIH3T3 and oncogenic H-RasV12-transforming NIH3T3 cells...............83
13. Effect of NGEF on the Angiogenesis in NIH3T3 and oncogenic H-RasV12-transforming NIH3T3 cells........86
14. Effect of NGEF on the Animal tumorigenesis ..89
15. Effect of NGEF on the Animal survival............93
16. Effect of NGEF on the Animal angiogenesis....95
17. Effect of NGEF on the Animal micrometastasis.97
IV. DISCUSSION............................................................99
V. REFERENCE...........................................................105
KOREAN ABSTRACT...................................................133
- Degree
- Doctor
- Publisher
- 조선대학교
- Citation
- 김 홍 범. (2007). The study for the oncogenic effect of NGEF.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/7018
http://chosun.dcollection.net/common/orgView/200000235922
- Appears in Collections:
- General Graduate School > 4. Theses(Ph.D)
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