臺灣博碩士論文加值系統

背景
微陣列晶片及基因體表現分析的進展提供同時研究數千基因表現的平台,也是探討腫瘤基因,傳導途徑變化上的利器. 雖然許多微陣列晶片數據已被發表並成為公開資訊, 關於鱗狀上皮食道癌的整合性微陣列晶片分析仍少有報告, 本計畫盼以整合性微陣列晶片分析探討鱗狀上皮食道癌的分子變化, 並研究其臨床意義.

方法
我們收集五個公開的微陣列晶片資料進行分析, 並發現了在鱗狀上皮食道癌中TPX2基因高度表現以及CRNN基因減少表現. 我們利用另一組核酸微陣列晶片驗證TPX2和CRNN的核酸表現, 並以免疫組織化學染色在福馬林固定石蠟包埋組織中驗證蛋白質的表現. 本計畫亦探討TPX2和CRNN表現的臨床病理相關性和預後意義. 在鱗狀上皮食道癌細胞中, 我們更進一步研究TPX2表現對其細胞生長的影響.

結果
免疫組織化學染色顯示TPX2表現增加和CRNN表現減少可分別在90.6%及77.7%的鱗狀上皮食道癌中發現. TPX2高度表現在單變量及多變量生存分析均是一個顯著的預後因子(HR = 1.802, p = 0.037). 在鱗狀上皮食道癌細胞中, 減低TPX2表現將造成細胞增殖的抑制, 細胞群落形成能力的下降. 此外, CRNN的表現下降與較深腫瘤浸潤深度(p = 0.002), 較嚴重淋巴結轉移(p = 0.014)和較長的腫瘤長度(p = 0.037)相關. CRNN呈陰性表現的病人也比CRNN呈陽性表現的病人有較差的整體存活率(p = 0.006). 在多變量分析中, CRNN表現低下(HR = 1.464, p = 0.047), 淋巴結轉移和遠處轉移都是預後不良的顯著指標.

結論
我們發現鱗狀上皮食道癌中TPX2基因高度表現以及CRNN基因減少表現.而TPX2的表現與鱗狀上皮食道癌細胞生長與病人的不良預後有關. CRNN的表現缺少亦與腫瘤惡性程度和不良預後相關.

Background
The advent of microarray technology and genome-wide expression profiling allows simultaneous expression analysis of thousands of genes, which provides a powerful tool for studying gene and pathway expression changes in the development of malignancies. Although an enormous amount of microarray data has been collected by various groups and deposited in public repositories, integrated microarray analysis of esophageal squamous cell carcinoma (ESCC) is lacking. We aim to study the molecular and genetic changes involved in ESCC pathogenesis with integrated microarray analysis and investigate their clinical relevance.
Methods
Five publicly available microarray datasets were collected for integrated microarray analysis. We identified TPX2 as one of the major up-regulated genes and CRNN gene as one of the major down-regulated genes in ESCC. The up-regulated expression of TPX2 and down-regulated expression of CRNN were validated at the nucleic acid level by cDNA microarray and at the protein level by immunohistochemical (IHC) stains in formalin-fixed paraffin-embedded (FFPE) specimens. The clinicopathological relevance and prognostic significance of TPX2 and CRNN expressions were explored. The impact of TPX2 expression was also accessed in ESCC cancer cells.
Results
Immunohistochemical staining revealed TPX2 overexpression and loss of CRNN expression in 90.6% and 77.7% of ESCC tumors, respectively. High TPX2 expression was a significant prognostic factor for overall and disease-free survival in univariate analysis and remained an independent prognostic factor in multivariate analysis (Hazrad ratio (HR) = 1.802, p = 0.037). TPX2 knockdown cells also showed inhibited cellular proliferation in growth curve studies and formed fewer colonies in the clonogenic assay. In addition, loss of CRNN protein expression was associated with advanced tumor invasion depth (p = 0.002), advanced nodal involvement (p = 0.014), and longer tumor length (p = 0.037). Patients with negative CRNN expression had worse overall survival than those with positive expression (p = 0.006). In multivariate analysis, negative CRNN expression, along with nodal involvement and distant metastasis, remained a significant prognostic factor for poor outcome (HR: 1.464, p = 0.047).
Conclusions
Up-regulation of TPX2 and down-regulation of CRNN expression in ESCC are demonstrated in the integrated microarray analysis. Furthermore, TPX2 expression is associated with cell proliferation and poor prognosis among ESCC patients. Loss of CRNN expression is correlated with tumor aggressiveness and poor survival in ESCC patients.

1. English Abstract----------------------------------------------------------------------- 5

2. Chinese Abstract--------------------------------------------------------------------- 7

3. List of Abbreviations----------------------------------------------------------------- 9

4. Introduction--------------------------------------------------------------------------- 10
4.1 Background – Previous clinical studies on esophageal cancer in
Taipei Veterans General Hospital-------------------------------------------- 10
4.2 Background - Previous immunohistochemistry studies on
esophageal cancer in Taipei Veterans General Hospital------------- 11
4.3 Microarray studies in esophageal cancer-------------------------------- 13
4.4 Aims of this study--------------------------------------------------------------- 17

5 Materials and Methods------------------------------------------------------------ 18
5.1 Experimental design and workflow----------------------------------------- 18
5.2 Microarray dataset collection and analysis------------------------------- 18
5.3 Verification of mRNA expression by cDNA microarray----------------- 19
5.4 Verification of protein expression by immunohistochemical stains- 20
5.5 Cell lines and plasmids-------------------------------------------------------- 22
5.6 Western blot analysis---------------------------------------------------------- 23
5.7 Functional characterization of TPX2 gene in cell lines----------------- 23
5.7.1 Wound healing assay-------------------------------------------------- 23
5.7.2 Chemosensitivity assay----------------------------------------------- 23
5.7.3 Cell proliferation assay------------------------------------------------ 24
5.7.4 Clonogenic assay------------------------------------------------------ 24
5.7.5 Cell cycle analysis------------------------------------------------------ 25
5.8 Statistical analysis-------------------------------------------------------------- 25

6 Results-------------------------------------------------------------------------------- 27
6.1 Analysis of publicly available microarray datasets---------------------- 27
6.2 AURKA and TPX2-------------------------------------------------------------- 27
6.2.1 AURKA and TPX2 as targeted up-regulated genes------------ 27
6.2.2 Verification of AURKA and TPX2 mRNA expression----------- 28
6.2.3 Verification of AURKA and TPX2 protein expression---------- 29
6.2.4. Impact of TPX2 expression on ESCC cell lines---------------- 30
6.2.4.1 Wound healing assay ------------------------------------- 31
6.2.4.2 Chemosensitivity assay-------------------------------------- 31
6.2.4.3 Cell proliferation assay--------------------------------------- 31
6.2.4.4 Clonogenic assay---------------------------------------------- 32
6.2.4.5 Cell cycle analysis--------------------------------------------- 32
6.2.4.5.1 Flow cytometric analysis----------------------------- 32
6.2.4.5.2 Expression of cyclins and CDKs------------------- 32
6.3 CRNN------------------------------------------------------------------------------ 32
6.3.1 CRNN as targeted down-regulated genes----------------------- 32
6.3.2 Verification of CRNN mRNA expression-------------------------- 33
6.3.3 Verification of CRNN protein expression-------------------------- 33
6.3.4 Clinical relevance of CRNN protein expression----------------- 35


7 Discussion---------------------------------------------------------------------------- 36
7.1 Integrated microarray analysis----------------------------------------------- 36
7.2 The physiological role of AURKA and TPX2------------------------------ 38
7.3 The role of AURKA and TPX2 in cancer formation--------------------- 38
7.4 The role of AURKA and TPX2 in ESCC----------------------------------- 40
7.5 The physiological role of CRNN function---------------------------------- 42
7.6 The role of CRNN in cancer formation------------------------------------- 43
7.7 The role of CRNN in ESCC--------------------------------------------------- 44

8 Conclusions-------------------------------------------------------------------------- 46

9 Perspectives ------------------------------------------------------------------------ 47

10 References--------------------------------------------------------------------------- 50

11 Tables and Figures----------------------------------------------------------------- 65
Table 1---------------------------------------------------------------------------------- 66
Table 2---------------------------------------------------------------------------------- 67
Table 3---------------------------------------------------------------------------------- 68
Table 4---------------------------------------------------------------------------------- 70
Table 5---------------------------------------------------------------------------------- 71
Table 6---------------------------------------------------------------------------------- 72
Table 7---------------------------------------------------------------------------------- 73
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Table 10-------------------------------------------------------------------------------- 76
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Table 12-------------------------------------------------------------------------------- 78
Table 13-------------------------------------------------------------------------------- 80
Figure 1--------------------------------------------------------------------------------- 81
Figure 2--------------------------------------------------------------------------------- 82
Figure 3--------------------------------------------------------------------------------- 83
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Figure 9--------------------------------------------------------------------------------- 89
Figure 10------------------------------------------------------------------------------- 90
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Figure 13------------------------------------------------------------------------------- 93
Figure 14------------------------------------------------------------------------------- 94
Figure 15------------------------------------------------------------------------------- 95
Figure 16------------------------------------------------------------------------------- 96
Figure 17------------------------------------------------------------------------------- 97
Figure 18------------------------------------------------------------------------------- 98
Figure 19------------------------------------------------------------------------------- 99

12 Publications-------------------------------------------------------------------------- 99

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