臺灣博碩士論文加值系統

　　癌症轉移是一連串包含基因改變的複雜的序列事件，包括週邊組織侵襲、穿透至血液或淋巴組織中、定位在遠端組織的血管內皮細胞上、穿出血管並引起血管新生作用，最後在遠端轉移組織長成次發的腫瘤。MTA1( metastasis- associated protein 1)首先是從老鼠轉移乳腺腫瘤cDNA庫(cDNA library from rat metastatic mammary adenocarcinoma)中首先選殖出來，並發現MTA1的mRNA表現量在老鼠乳癌腫瘤和人類乳癌細胞中有上升的傾向。先前的幾個研究顯示MTA1 mRNA表現量似乎也和具侵襲性的人類腫瘤有關，包括結腸直腸癌、胃癌食道癌及胸腺瘤。MTA1剪裁變異型(MTA1s)是MTA1經過選擇性接合作用（alternative splicing）去除外顯子-14的47對核苷酸，造成框移突變(frame shift mutation)產生了一個提前終止訊號(stop codon)而得到MTA1 C端截切產物。值得注意的是MTA1s缺乏核內定位信號（nuclear localization signal），MTA1s能和雌激素受體交互作用，並將雌激素受體捕捉在細胞質中。最近研究顯示，MTA1s過度表現在乳癌細胞中可能與anchorage-independent及tumorigenic的表現型有關。首先，在本研究中，我們測試MTA1及MTA1s與乳癌轉移及侵襲相關之假說，我們利用即時偵測同步定量之聚合酶連鎖反應器（real-time polymerase chain reaction），定量乳癌組織中MTA1及MTA1s之表現量。我們發現在病人的腫瘤組織中，在淋巴結轉移≧2（n=13）的病人其腫瘤組織的MTA1s/ MTA1比值比鄰近週邊正常組織來的高，並且也有顯著差異(p=0.041)。其次，我們利用EGFP-N1、EGFP- MTA1及EGFP- MTA2瞬時轉染老鼠胚胎細胞NIH3T3並進行細胞移行及細胞侵襲性分析。在細胞移行實驗中，EGFP-MTA1、EGFP-MTA2分別比EGFP-N1控制組高1.4及1.3倍;在細胞侵襲試驗(n=6)中，EGFP- MTA1，EGFP-MTA2分別比EGFP- N1控制組高1.6及 1.4倍。我們結果顯示乳癌患者腫瘤組織MTA1s/ MTA1比值越高，其臨床的表現越不佳。我們認為定量分析MTA1和MTA1s有潛力成為乳癌轉移的生物指標。

　Metastasis is a complex series of events that involves several gene products, including those for the invasion and detachment of neoplastic cells from the primary tumor, penetration into blood and lymphatics, arrest at distant sites by adhesion to endothelial cells, extravasation, induction of angiogenesis, invasion from host antitumor responses, and growth at metastatic sites. The metastasis-associated gene 1 (mta1) was identified by differential cDNA screening using cell lines derived from highly metastatic mammary adenocarcinomas. Enhanced expression of mta1 mRNA was also found in a variety of other human cancerous tissues and carcinoma cell lines, including in colorectal, gastric, or oesophageal carcinomas and thymoma. A naturally occurring variant of MTA1 was recently discovered. This variant, named MTA1s (for short version of MTA1), is a C-terminal truncated form of MTA1, generated by alternative splicing at a cryptic splice site in exon 14, resulting in a deletion of 47 base-pair nucleotides. The underlying mechanism of MTA1s-mediated repression of the estrogen receptor (ER) transactivation function involves nuclear exclusion and sequestration of ER in the cytoplasm. Results of studies involving MTA1s overexpression suggest a role of MTA1s in conferring anchorage independent and tumorigenic phenotypes in breast cancer cells. In this study, we tested the hypothesis that the expression of MTA1 and MTA1s was correlated with the invasive and metastatic potential of breast cancer. First, we used quantitative real-rime PCR to detect mRNA expression levels of mta1 and mta1s in breast cancer tissue. The result revealed that mta1s/mta1 mRNA ratio was significantly higher in metastasis≧2 breast cancer tissue than adjacent normal tissue (p=0.041). Second, we also used migration assay and invasion assay with Boyden chamber and transwell to analysis the association between the level of mta1 and cancer metastasis. We found that transient transfection of NIH3T3 by EGFP-MTA1, EGFP-MTA2, and EGFP-N1 caused a 1.4-fold and 1.3-fold increased migration and 1.6-fold and 1.4-fold increased invasion, compared to the corresponding EGFP-N1 cells. Our results suggest that the patients with breast cancer that have a more aggressive clinical behavior may have higher mta1s/mta1 mRNA ratio. We conclude that expression of mta1 and mta1s may act as a potential biological marker in breast cancer progression.

目錄（Index）
中文摘要 I
英文摘要 II
誌謝 IV
目錄 V
表/圖/附錄圖表目錄 XI
縮寫 XIII
藥品、儀器 XIV

第一章 緒論（Introduction） 1
一. 乳房的解剖結構 3
二. 乳癌的流行病學 3
三. MTA (Metastasis-associated gene) Family 4
1. MTA蛋白之功能 4
2. MTA蛋白之表現及調控 6
3. MTA1 (Metastasis-associated protein 1) 7
4. MTA1 和癌症之間的關係 8
5. MTA1的基因衍生物: MTA1剪裁變異型 9
四. 雌激素受體 11
1. 雌激素受體之功能 12
1. 雌激素受體之分型 13
(1) 雌激素受體-alpha�� 13
(2) 雌激素受體-bata 13
(3)外顯子缺失之雌激素受體-alpha mRNA變異型 13
(4)外顯子-3缺失（ER alpha E3D） 14
(5)外顯子-5缺失（ER alpha E5D） 14
(6)外顯子-7缺失（ER alpha E7D） 15
五. 研究方向 16
1. MTA1 (metastasis-associated protein 1) 和MTA1s
(MTA1 short form)與乳癌轉移的關係 16
2. 分析MTA1改變細胞表面黏著因子而造成腫瘤轉移 16


第二章 目標（Aim） 18
一. 研究目的 19
二. 研究設計 19
三. 研究對象 20
四. 資料處理與統計分析 20
五. 細胞移行試驗(Cell migration assay)分析MTA1和腫瘤
轉移的關係 20

第三章 材料與方法（Materials and Methods） 21
壹. 檢體收集與建立資料 24
1. 檢體採集與處理 24
2. 資料處理 24
貳. 獲取MTA1及MTA1剪裁變異型之片段 25
一. 細胞培養 25
1. 細胞繼代培養 25
2. 細胞冷凍保存 27
3. 冷凍細胞之再培養 28
二.細胞Total RNA 的抽取 29
三. 反轉錄聚合酶連鎖反應 31
1. 反轉錄作用 31
2. 聚合酶連鎖反應 32
3. 聚合酶連鎖反應產物純化 33
參.構築標準質體 35
1. 勝任細胞的製備 35
2. A-tailing 36
3. 接合作用 37
4. 轉型至勝任細胞中 38
5. 小量質體萃取 39
6. DNA定序 40
肆. 定量組織中MTA1及MTA1剪裁變異型表現量 42
1. 組織Total RNA的抽取 42
2. RNA電泳 43
3. 反轉錄作用 44
4. 即時性聚合酶連鎖反應 45
伍.定量組織中GAPDH mRNA表現量 46
1. 反轉錄作用 46
2. 即時性聚合酶連鎖反應 47
陸.統計分析 48
1. 曼-惠特尼檢定（Mann-Whitney Test） 48
2. 魏可遜配對組符號等級檢定
（Wilcoxon Signed Ranks Test） 49
3. 史皮爾曼相關係數檢定
（Spearman’s rho Correlation Test） 50
4. 線性迴歸分析（Linear Regression Analysis） 51
柒.分析MTA1改變細胞表面黏著因子表現差異而
造成腫瘤的轉移 52
1. 瞬時轉染細胞株 (Tansient transfection) 52
2. 細胞趨化性試驗 (Cell Migration Assay) 53
3. 細胞侵襲試驗 (Cell Inversion Assay) 54

第四章 結果（Result） 56
一. 構築標準質體 59
二.組織Total RNA的抽取 59
三. 即時性聚合酶連鎖反應-定量分析雌激素受體mRNA變異
型之轉錄活性 59
Ⅰ.定量分析MTA1和MTA1剪裁變異型mRNA變異型之轉錄活性 59
1. 檢量標準區曲線的建立 59
(1) MTA1 60
(2) MTA1剪裁變異型（MTA1s） 60
2. 引子對專一性 60
(1) MTA1 60
(2) MTA1剪裁變異型（MTA1s） 61
Ⅱ. 定量GAPDH mRNA之轉錄活性 61
四. MTA1和MTA1剪裁變異型mRNA表現量與台灣地區乳癌
的相關性 62
(1) MTA1 63
(2) MTA1剪裁變異型（MTA1s） 65
(3) MTA1剪裁變異型相對於MTA1之比值 （MTA1s/MTA1） ����
(4)MTA1和MTA1剪裁變異型在遠端正常組織與腫瘤組織之差異 68 ����
五. MTA1和MTA1剪裁變異型與雌激素受體基因型和雌激素受
體變異型mRNA表現量之相關性 69
六. 分析MTA1改變細胞表面黏著因子表現差異而造成腫瘤
的轉移 69
(1) 細胞移行試驗 (Cell Migration Assay) 69
(2) 細胞侵襲試驗 (Cell Inversion Assay) 70

第五章 討論（Discussion） 71
一. MTA1 mRNA表現量與文獻之比較 72
二. MTA1剪裁變異型（MTA1s）mRNA表現量及MTA1剪裁
變異型相對於MTA1之比值（MTA1s/MTA1）與台灣
地區乳癌的相關性 73
三. MTA1剪裁變異型與雌激素受體變異型mRNA表現量
之相關性 74
四. 細胞移行試驗(Cell migration assay)分析MTA1
和腫瘤轉移的關係 75
五. 結語 76

參考文獻（References） 77

表/圖（Tables/Figures） 86
附錄圖表（Appendix） 100
自述（Author） 106

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