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系統識別號 U0026-2607201310230800
論文名稱(中文) 探討在肝癌細胞中CPAP 與HBx 蛋白的相互關係
論文名稱(英文) Studying the relationship between CPAP and HBx in hepatocellular carcinoma
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 101
學期 2
出版年 102
研究生(中文) 許魯信
研究生(英文) Lu-Shin Hsu
學號 S26001046
學位類別 碩士
語文別 英文
論文頁數 50頁
口試委員 指導教授-洪良宜
口試委員-張文昌
口試委員-陳炳焜
口試委員-顏家瑞
中文關鍵字 肝細胞癌  CPAP  B 型肝炎病毒X 蛋白  NF-kB 
英文關鍵字 HCC  CPAP  HBx  NF-kB 
學科別分類
中文摘要 慢性發炎為導致肝癌的其中一個原因,而造成肝細胞慢性發炎的可能原因為肝炎病毒、酗酒或其它有毒的化學物質。持續活化的nuclear factor-kB (NF-kB)為參與肝臟細胞慢性發炎的重要因子。另一方面,於中心體功能上扮演重要角色的CPAP (Centrosomal protein P4.1-associated protein) 蛋白在先前的報導中被認為是NF-B 的轉錄共同活化者(transcriptional co-activator)。此外,過去文獻報導指出B 型肝炎病毒中的 X 蛋白 (HBx) 可增加NF-B 的活性。在本論文,我們發現GFP-CPAP 可協同性增加HBx 所調控的NF-kB 轉錄活性。利用siRNA抑制CPAP 表現,則減弱了此作用。利用免疫沉澱法(immunoprecipitation assay)顯示CPAP 與HBx 有交互作用。有趣的是,HBx 可透過CREB (cyclic adenosine monophosphate response element-binding protein) 來增加CPAP 的啟動子(promoter) 活性並造成CPAP 表現量的增加。此外,我們也發現在CPAP 大量表現的肝癌細胞株中,細胞增生能力有增加的情形。總結來說,我們的結果指出CPAP 可與HBx 合作來增加NF-kB 的活性,而這可能會導致肝癌的生成。
英文摘要 Hepatocellular carcinoma (HCC) is a type of tumor that results from chronic inflammation triggered by hepatotropic viruses, alcoholism or other toxic chemicals. Constitutive activation of nuclear factor-kB (NF-kB) is an important event involved in chronic inflammation of liver. CPAP (Centrosomal protein P4.1-associated protein), which plays an important role in centrosomal functions, is previously
identified as a transcriptional co-activator of NF-kB. In addition, it was reported that the HBV X protein (HBx) of hepatitis B virus (HBV) can enhance the activation of NF-kB. In this study, we demonstrated that overexpression of GFP-CPAP synergistically increases the HBx-mediated NF-kB transcriptional activity, whereas knockdown expression of CPAP by siRNA attenuates this effect. Immunoprecipitation assay showed that CPAP interacts with HBx. Interestingly,
HBx enhances the promoter activity of CPAP may be through the interaction with cyclic adenosine monophosphate response element-binding protein (CREB). Stably expression of CPAP in HCC cell lines increased the cell proliferation ability. Taken together, our results indicate that CPAP can cooperate with HBx to increase the NF-kB activity, which may contribute to hepatocarcinogenesis.
論文目次 Abstract in Chinese I
Abstract II
Acknowledgments III
Table of Contents IV
List of Figures VII
List of Appendices VIII
List of Abbreviations IX

Chapter 1. Introduction 1
1. Hepatitis B virus (HBV) and hepatocellular carcinoma
(HCC) 1
2. HBV X protein(HBx) 1
3. HBx and cAMP response element-binding protein (CREB) 3
4. Centrosomal protein P4.1-associated protein (CPAP) 3
5. Nuclear factor-kB (NF-kB) and inflammation 3
6. Research motivation and specific aims 4

Chapter 2. Materials and methods 6
1. Cell culture and DNA plasmid transfection 6
2. Total RNA isolation, reverse transcription (RT)-PCR, and
real-time PCR 6
3. Immunoblotting and antibodies 7
4. Small interfering RNA transfection 8
5. Promoter construct and reporter assay 8
6. NF-kB lucuferase reporter gene assay 9
7. Co- immunoprecipitation assay (Co-IP) 10
8. Cell proliferation assay 11

Chapter 3. Results 12
A. CPAP increases the transcriptional activity of NF-kB 12
A-1. CPAP increases NF-kB transcriptional activity upon different inflammatory cytokines stimulus 12
A-2. Knockdown expression of CPAP decreases TNFa-induced NF-kB transcriptional activity 12
A-3. CPAP enhances NF-B transcriptional activity through increasing the phosphorylation of NF-kB and the degradation of IkBa 13
B. CPAP interacts with HBx and is required for HBx-induced NF-kB activation 13
B-1. CPAP is required for HBx-induced NF-kB activation 14
B-2. CPAP interacts with HBx 14
C. HBx increases the expression of CPAP 14
C-1. The expression level of CPAP in HBx-expressing HCC cells is higher than that in parental cells 14
C-2. HBx increases the promoter activity of CPAP 15
D. HBx cooperates with CREB to enhance CPAP promoter activity 15
D-1. HBx regulates CPAP promoter activity through CREB binding to the CPAP promoter -86 to -79 bp region 15
D-2. Dose effect of HBx on activating CPAP promoter
activity 17
E. CPAP increases the protein stability of HBx 17
F. CPAP cooperates with HBx to regulate the cell proliferation ability of HCC 18
F-1. Generation of GFP-CPAP stably expressed Hep3B cell lines 18
F-2. GFP-CPAP increases the cell proliferative ability 18
F-3. HBx and CPAP cooperates to increase the cell proliferation ability 19

Chapter 4. Discussions 20
1. The possible mechanism of HBx in trans-activating CPAP 20
2. How does centriolar protein CPAP enhances HBx protein stability ? 21
3. CPAP SUMOlylation may involve in the protein-protein interaction with HBx protein 22
4. HBx increases CPAP expression through regulating CPAP promoter activity but not in a dose dependent manner 22

Chapter 5. Conclusions 24

References 25
Figures 28
Appendices 43
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