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研究生：	陳若瑜 Chen, Ruo-Yu
論文名稱：	CPAP參與在肝癌形成以及轉移之研究 Studying the role of CPAP in hepatocarcinogenesis and metastasis
指導教授：	洪良宜 Hung, Linag-Yi
學位類別：	博士 Doctor
系所名稱：	生物科學與科技學院 - 生物科技與產業科學系 Department of Biotechnology and Bioindustry Sciences
論文出版年：	2019
畢業學年度：	108
語文別：	英文
論文頁數：	92
中文關鍵詞：	中心體蛋白、惡性肝癌、轉移
外文關鍵詞：	CPAP, HCC, Metastasis
相關次數：	點閱：95 下載：1
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我們先前發現，中心體蛋白Centrosomal P4.1-associated protein (CPAP)在肝癌細胞中會增加TNF-α誘導的NF-κB活化以及IL-6誘導的STAT3活化；在臨床分析，CPAP與血管入侵，術後復發以及腫瘤分級呈現正相關。因此，本研究想進一步探討CPAP在發炎誘導HCC形成以及惡化過程中所扮演的功能性角色。首先，當CPAP基因轉殖鼠處理DEN後，其ALT數值、肝臟細胞受損情況、促發炎因子表現以及NF-κB以及STAT3活化都會增加；CPAP也會增加DEN誘導的惡性肝腫瘤的生長。在臨床分析以及動物實驗結果看到，CPAP與肝癌腫瘤生長以及轉移是有正相關的。CPAP過度表現時也會提升腫瘤細胞爬行/入侵以及血管新生的能力。接著，在IL-6處理後，CPAP可以透過增加IL-6/STAT3活化進而調控細胞爬行和入侵的能力。在機制中，CPAP除了會增加STAT3的活化，亦會增加磷酸化的STAT3進入細胞核內。功能上，CPAP可以透過IL-6/STAT3/IL-8路徑來增強腫瘤細胞的血管新生能力；增加CD44的表現量；減少腫瘤抑制基因如PTEN以及MTSS1的表現。在帶有血管入侵特徵的HCC檢體中，CPAP mRNA表現與IL-8以及CD44 mRNA表現呈現正相關。這些結果表示CPAP是透過增加IL6/STAT3路徑來調控腫瘤細胞的惡化特性以導致肝癌轉移。

Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer death worldwide. HCC is also associated with chronic liver inflammation. Our previous studies indicated that overexpressed CPAP contributes to the inflammatory microenvironment of HCC by increasing the NF-κB and STAT3 transcriptional activities. However, the functional role of CPAP in inflammation-mediated hepaticarcinogenesis and HCC progression remains unclear. Here, we found that CPAP enhances the diethylnitrosamine (DEN)-induced HCC formation and increases the HCC malignant features via increasing the IL-6-mediated STAT3 activity. The expression level of CPAP is positively correlated with IL-8 and CD44 in HCC tissues with vascular invasion. Taken together, our study indicated that CPAP promotes the development and progression of HCC via enhancing the IL-6/STAT3 pathway.
Key words: CPAP, HCC, Metastasis

Chinese Abstract (中文摘要)............................... I
Abstract................................................ II
Acknowledgements........................................ IV
Table of Contents........................................ V
Contents of Tables....................................... X
Contents of Figures..................................... XI
Abbreviation List....................................... XV
Chapter 1 Introduction................................... 1
1-1	Inflammation is an important hallmark of cancer.. 1
1-2	Hepatocellular carcinoma (HCC) and its therapeutic efficiency............................................... 1
1-3	HCC and angiogenesis............................. 3
1-4	NF-κB and STAT3 are two major inflammatory signaling pathways in the development and progression of HCC...................................................... 4
1-5	Centrosomal P4.1-associated protein (CPAP)....... 5
1-6	CPAP contributes to the acitvation of TNF-α/ NF-κB in HCC................................................... 6
1-7	Research Objectives.............................. 8
Chapter 2 Materials and Methods.......................... 9
2-1	Materials........................................ 9
2-2	Methods......................................... 15
2-2-1	Low doses of DEN-induced acute liver inflammation and high doses of  DEN-induced animal model............. 15
2-2-2	Generation of albumin-CPAP transgenic mice and determination of the genotype........................... 15
2-2-3	Determination of ALT value...................... 16
2-2-4	Western blot analysis........................... 16
2-2-5	RNA extraction, reverse transcription and real-time PCR..................................................... 16
2-2-6	Cell culture.................................... 17
2-2-7	IL-6 and TNF-α treatment........................ 17
2-2-8	Collection of conditioned medium................ 17
2-2-9	Plasmid transfection and reporter gene assay.... 18
2-2-10	Fractionation of nuclear and cytosolic extracts. 18
2-2-11	Co-immunoprecipitation assay.................... 19
2-2-12	Migration / invasion assay...................... 19
2-2-13	HUVECs migration assay / Tube formation of HUVECs assay................................................... 20
2-2-14	siRNA and shRNA transfection.................... 20
2-2-15	Tumor metastasis RT2 PCR array.................. 20
2-2-16	The expression of CPAP in human cancers......... 21
2-2-17	Orthotopic mouse model of hepatocellular carcinoma............................................... 22
2-2-18	Mouse model of intrasplenic injection........... 22
2-2-19	Statistical analysis............................ 22
Chapter 3 Results....................................... 24
3-1	Overexpression of CPAP in mice liver enhances DEN-induced acute inflammatory response through NF-κB and STAT3 singaling............................................... 24
3-2	CPAP overexpression enhances DEN-indcured hepatocarcinogenesis.................................... 25
3-3	CPAP is involved in the growth and metastasis of HCC..................................................... 26
3-3-1	Overexpression of CPAP increases liver tumor growth and HCC metastasis...................................... 26
3-3-2	Overexpression of CPAP promotes HCC metastasis in an intra-splenic injection model........................ 27
3-4	Overexpression of CPAP increases malignant features of HCC cell through IL-6/STAT3 signaling................ 28
3-4-1	Overexpression of CPAP increases HCC cell migration and invasion............................................ 28
3-4-2	Overexpression of CPAP increases the malignant features of HCC cell through IL-6/STAT3 signaling....... 28
3-5	CPAP is requied for IL-6/STAT3 activation in HCC..................................................... 29
3-5-1	Overexpression of CPAP increases IL-6/STAT3 signaling transcriptional activity...................... 29
3-5-2	IL-6 increases the nuclear translocation of both CPAP and STAT3.......................................... 29
3-6 	CPAP increases the angiogenic ability of HCC cells via IL6/STAT3/IL-8 signaling, and is positively correlated with expression of IL-8 upon vascular invasion in HCC... 30
3-6-1	CPAP overexpressing HCC cells increases IL-6/STAT3-mediated recruitment and tube formation of HUVECs and angiogenesis in a matrigel plug tumor................... 30
3-6-2	IL-8 is a downstream effector of CPAP-mediated angiogenic ability in HCC cells......................... 31
3-6-3	Overexpression of CPAP increases the expression of IL-8 in plug tumors and liver tumor with lung metastasis.............................................. 32
3-6-4	There is a positive correlation between CPAP and IL-8 in HCC with vascular invasion...................... 32
3-7	CPAP increases the expression of STAT3 target genes involved in metastasis.................................. 33
3-7-1	Oeverexpression of CPAP increases other metastatic genes in HCC cells...................................... 33
3-7-2	A positive correlation was found between CPAP and two metastatic genes in HCC............................. 33
3-7-3	A negative correlation weas found between CPAP and tumor suppressor genes in HCC........................... 34
Chapter 4 Discussion.................................... 36
4-1	The effect of CPAP in inflammation-induced hepatocytes death and maintenance....................... 36
4-2	CPAP is involved in inflammation –mediated hepatocarcinogenesis.................................... 37
4-3	CPAP plays an important role in the development and progression of HCC...................................... 38
4-4	CPAP is required for the IL-6/STAT3-mediated malignant features of HCC............................... 39
4-5	CPAP enhances IL-6/STAT3 transcriptional activity in HCC.................................................. 39
4-6	CPAP contributes to angiogenesis via IL-6/STAT3/IL-8 signaling............................................. 40
4-7	The correlation between CPAP and IL8 in HCC with vascular invasion....................................... 41
4-8	Another functional role of CPAP in the malignant progression of HCC via IL6/STAT3 signaling.............. 41
4-9	The inhibitor of blocking CPAP and STAT3 interaction as a potential therapeutic strategy in HCC.. 43
References.............................................. 45
Tables.................................................. 51
Figures................................................. 55
Appendices.............................................. 88
Related paper Publication............................... 92

                                    

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校內：2023-08-01公開
校外：不公開電子論文尚未授權公開，紙本請查館藏目錄

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