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系統識別號 U0026-2407201413483700
論文名稱(中文) 腫瘤抑制蛋白質含雙色胺酸功能區氧化還原酶對細胞生長的調控
論文名稱(英文) The control of cell growth by tumor suppressor WWOX
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 102
學期 2
出版年 103
研究生(中文) 鄭慧卿
研究生(英文) Hui-Ching Cheng
學號 T36004055
學位類別 碩士
語文別 英文
論文頁數 74頁
口試委員 指導教授-徐麗君
口試委員-林以行
口試委員-詹明修
中文關鍵字 含雙色胺酸功能區氧化還原酶  腫瘤抑制蛋白質  細胞生長 
英文關鍵字 WOX1  tumor suppressor  cell growth 
學科別分類
中文摘要 人類WOX1基因位在第16對染色體易脆裂 (fragile) 的16q 23.3-24.1位點上,可轉錄轉譯出含雙色胺酸功能區氧化還原酶 (WW domain-containing oxidoreductase)。過去研究指出,在許多人類癌症,WOX1蛋白質表現量有明顯下降的情形,故WOX1一直被視為一種腫瘤抑制基因。在本研究中,我們使用WOX1基因剔除小鼠胚胎纖維母細胞 (mouse embryonic fibroblasts;MEFs) 來探討WOX1在調控細胞生長上的角色。有趣的是,我們的實驗結果發現,WOX1基因剔除的MEFs生長速度明顯比控制組MEFs快速,而且細胞周期分布情形也不相同;此外,WOX1基因剔除的MEFs、和控制組MEFs的細胞中,與調控細胞周期相關的蛋白質、以及cyclin-dependent kinase inhibitors的表現情形有顯著的差異。總結所有的實驗結果,我們的結果顯示WOX1在調控MEF細胞生長的機制中扮演重要的角色。未來,我們將繼續剖析WOX1在調控MEF細胞生長變化中的分子機制,以及WOX1在細胞生理上所扮演的角色給予定義。藉由對WOX1在細胞生長調控機制的瞭解與描述,在腫瘤形成學裡將針對WOX1的角色提供更嶄新的知識。
英文摘要 WW domain-containing oxidoreductase, designated WOX1, is encoded by WOX1 gene. Human WOX1 gene locates at a common fragile site on chromosome 16q 23.3-24.1. Because downregulation of WOX1 protein expression has been found in many types of human cancers, WOX1 is considered as a tumor suppressor gene. In this study, we generated gene knockout mouse embryonic fibroblasts (MEFs) to explore the potential role of WOX1 in the regulation of cell growth. Interestingly, accelerated cell growth rate and altered cell cycle distribution were detected in WOX1 gene knockout cells, as compared with the control MEFs. The expression profiles of cell cycle-related proteins and cyclin-dependent kinase inhibitors were significantly different in WOX1 gene knockout and control MEFs. In conclusion, we show here that WOX1 controls MEF cell growth. The molecular mechanism by which WOX1 regulates the phenotypic changes of MEFs and the physiological role of WOX1 in cells will be further explored. Delineating the regulation of cell growth by WOX1 will provide a novel impact on the role of WOX1 in tumorigenesis.
論文目次 中文摘要 I
English Abstract II
Acknowledgement III
Abbreviations and Symbols V
Contents VI
Figure Index IX
Introduction 1
WW domain-containing oxidoreductase 1
WWOX functions as a tumor suppressor 1
Cell Cycle 2
Cell cycle regulation 3
CKIs 3
p21Cip1 3
p27Kip1 4
Cellular senescence 5
Materials and Methods 6
A. Materials 6
A-1 Cell lines 6
A-2 Chemicals and reagents 6
A-3 Kits 9
A-4 Drugs 9
A-5 Antibodies 9
A-6 Forward and reverse PCR primers 10
A-7 Consumables 11
A-8 Instruments 12
B. Methods 15
B-1 Cell culture 15
B-2 Senescence β-galactosidase staining 15
B-3 Flow cytometry: PI staining 16
B-4 Flow cytometry: BrdU incorpration staining 17
B-5 Cell death: Annexin V staining 18
B-6 Protein extraction 19
B-7 Quantification and adjustment of proteins 21
B-8 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting 21
B-9 RNA extraction 23
B-10 Reverse transcription polymerase chain reaction (RT-PCR) 25
B-11 Polymerase chain reaction (PCR) 26
B-12 Real-time polymerase chain reaction (Real-time PCR) 29
B-13 Plasmid DNA purification 30
B-14 Transfection of cells by electroporation 32
B-15 Promoter activity reporter assay 33
B-16 Immunofluorescence staining 34
B-17 Micronucleus assay 35
B-18 Cell fractionation 36
B-19 ChIP assay 37
Results 44
WWOX regulates cell growth and senescence 44
Increased cell cycle progression and cell death in late-passage Wwox-/- MEFs 44
Accelerated degradation of p27Kip1 protein in late-passage Wwox-/- MEFs 45
Down-regulation of p21Waf1/Cip1 mRNA and protein expression in late-passage Wwox-/- MEFs 46
Upregulation of WWOX protein expression in response to DNA damages in SCC-15 cells 47
Increased DNA damage in late-passage Wwox-/- MEFs 47
Increased nuclear p53 protein fails to bind to p21 promoter in late-passage Wwox-/- MEFs. 48
Discussion 49
The role of WWOX in controlling cell growth and senescence 49
The dual role of WWOX in mediating cell survival and death 49
The role of WWOX in DNA damage response 50
WWOX in p53/p21-dependent senescence pathway 50
The relationship between WWOX and p53 51
WWOX in p53-independent senescence pathway 52
Clinical correlation between WWOX and cell growth regulation 52
Conclusion 53
References 54
Figures 60
Appendixes 72
Resume 74

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