Tamoxifen (TAM)具有拮抗雌性激素活性，主要用於治療ER+乳癌患者。雖然研究證實使用TAM輔助治療可顯著降低乳癌復發率和死亡率，但仍有1/3接受TAM治療之乳癌患者會產生復發情形。TAM抗藥性是目前乳癌治療所面臨問題之一，若能了解與TAM抗藥性相關基因之表現情形，將有助於提升TAM對於乳癌患者之治療成效。因此，本研究利用基因網絡分析，探究誘發乳癌細胞產生TAM 抗藥性之關鍵基因。本研究從ArrayExpress匯集98個非TAM抗藥性乳癌細胞株及97個TAM抗藥性乳癌細胞株之微陣列資料，得知TAM抗藥性乳癌細胞對於非TAM抗藥性乳癌細胞之差異性表現基因(differentially expressed genes; DEGs)。以NetworkAnalyst分析DEGs並建立基因模組，進一步以Cytoscape分析各基因模組之基因網絡，整合建構出TAM抗藥性乳癌細胞之可能基因網絡和路徑。基因網絡結果顯示ADM/FOS/ESR1/IGF-1/HIF-1α/ABCG2路徑為TAM抗藥性乳癌細胞之關鍵基因路徑，以ROC曲線(receiver operating characteristic; ROC curve)評估發現此六個基因能為預測臨床乳癌病患預後之分子標誌。本研究亦利用非TAM抗藥性人類乳癌細胞(MCF-7)及TAM抗藥性人類乳癌細胞(MCF-7/TamR7)驗證此乳癌TAM抗藥性基因網絡，發現adrenomedullin (ADM)、Fos proto-oncogene, AP-1 transcription factor subunit (FOS)、estrogen receptor 1 (ESR1)、insulin like growth factor 1 (IGF-1)、hypoxia inducible factor 1 alpha subunit (HIF-1α)及ATP binding cassette subfamily G member 2 (ABCG2)基因表現在MCF-7/TamR7細胞均較MCF-7細胞高，轉染siADM後會降低ADM下游FOS、ESR1、IGF-1、HIF-1α及ABCG2基因表現，以及增加細胞凋亡(apoptosis)情形，進而降低MCF-7/TamR7細胞存活率。本研究發現ADM/FOS/ESR1/IGF-1/HIF-1α/ABCG2路徑為TAM抗藥性乳癌細胞之關鍵基因路徑之一，降低ADM基因具有抵禦乳癌細胞產生TAM抗藥性之潛力。
關鍵字：乳癌、tamoxifen、抗藥性、基因網絡分析、adrenomedullin

Abstract
Tamoxifen (TAM) is an antagonist of estrogen receptor (ER) usually used to treat patients with ER+ breast cancer. Adjuvant therapy with TAM has been obviously shown to decrease the recurrence and mortality of breast cancer. However, recurrence occurs in one third of breast cancer patients within 5 years after TAM therapy. Resistance to TAM is one of obstacles in breast cancer treatment. A better understanding of altered gene expressions associated with TAM resistance would solve this problem. Thus, this study performed a meta-analysis of gene networks to investigate whether TAM resistance in breast cancer is induced underlying the alteration of gene expressions.
This study collected 98 microarray samples of human breast cells non-resistant to TAM and 97 microarray samples of human breast cells resistant to TAM from ArrayExpress to identify the differentially expressed genes (DEGs) for TAM resistance in breast cancer. The module genes of DEGs conducted from NetworkAnalyst were further illustrated by Cytoscape to explore regulatory pathways in the integrated gene network of TAM resistance in breast cancer. The results of gene-network analysis showed that ADM/FOS/ESR1/IGF-1/HIF-1α/ABCG2 pathway was associated with TAM resistance in breast cancer. The results of ROC analysis revealed that ADM、FOS、ESR1、IGF-1、HIF-1α and ABCG2 genes could as biomarkers for predicting prognosis in TAM therapy for breast cancer. Furthermore, the result of gene-network analysis was validated in human breast cancer MCF-7 cells non-resistant to TAM and in human breast cancer MCF-7/TamR7 cells resistant to TAM. The significantly higher expressions of ADM、FOS、ESR1、IGF-1、HIF-1α and ABCG2 genes were found in MCF-7/TamR7 cells as compared with MCF-7 cells. Additionally, the cell viability was decreased accompanied with cell apoptosis after MCF-7/TamR7 cells were transfected with siADM to knockdown ADM and attenuate the expression of its down-stream genes. In conclusion, this study revealed that ADM/FOS/ESR1/IGF-1/HIF-1α/ABCG2 pathway is one of key points in breast cancer cells resistant to TAM, and reduced ADM gene has the potential to resist TAM resistance to breast cancer cells.

Keywords: breast cancer, tamoxifen, drug resistance, gene network, adrenomedullin

目錄
一、 緒論 1
二、文獻回顧 2
2.1 乳癌背景 2
2.2 乳癌危險因子 3
2.3 乳癌分期 4
2.4 乳癌分類 4
2.5 TAM作用機轉與臨床應用 5
2.7 TAM抗藥性相關研究 10
2.8 乳癌TAM抗藥性相關之關鍵基因介紹 11
2.9 其他乳癌治療藥物之抗藥性相關研究 14
2.10 Microarray與乳癌抗藥性相關研究 15
三、研究目的 18
四、研究材料與方法 19
4.1 收集Microarray數據集 19
4.2 微陣列數據資料之前置處理 20
4.3 差異性基因及基因模組分析 20
4.4 基因功能網絡圖及基因交互作用網絡圖 21
4.5 評估關鍵基因之預測效度 21
4.6 細胞來源及配製細胞培養液 22
4.7 細胞繼代 23
4.8 細胞計數 23
4.9 細胞毒性分析 24
4.10 RNA萃取 24
4.11 RNA反轉錄為cDNA 25
4.12 定量聚合酶鏈鎖反應定量基因表現 25
4.13 細胞轉染實驗 26
4.14 細胞凋亡實驗 27
4.15 數據統計 27
5.1 建立TAM抗藥性乳癌細胞株基因模組之基因功能網絡及基因交互作用網絡 29
5.2 建立TAM抗藥性乳癌細胞株之高度連結性基因網絡及關鍵基因路徑 31
5.3 乳癌細胞TAM抗藥性關鍵基因之效度評估 32
5.4 乳癌細胞之抗藥性細胞存活率評估 34
5.5 乳癌細胞之關鍵性基因表現 35
5.6 乳癌細胞轉染siADM之關鍵性基因表現 37
5.7 乳癌細胞轉染siADM後對於TAM抗藥性之細胞存活情形 40
5.8 乳癌細胞轉染siADM前後之細胞凋亡情形 41
六、討論 44
6.1乳癌細胞產生TAM抗藥性藉由影響上皮細胞間質轉化、細胞訊息傳遞路徑及細胞死亡功能 44
6.2 活化ADM/FOS/ESR1/IGF-1/HIF-1α/ABCG2 路徑增加乳癌細胞抗藥性 46
6.3透過siADM下調ADM/FOS/ESR1/IGF-1/HIF-1α/ABCG2路徑可促使細胞凋亡及降低乳癌細胞抗藥性 50
七、結論 54
參考文獻 55















圖目錄
圖1 2016年台灣兩性前十大癌症死因死亡率 2
圖2 2016年美國兩性前十大癌症死亡人數及死亡分率 2
圖3 給予TAM治療一年、二年及五年之復發和死亡率情形 6
圖4 TAM的正向與負向效應 7
圖5 抗藥性機制示意圖 9
圖6 篩選人類乳癌細胞TAM抗藥性相關之基因微陣列數據集之流程圖 20
圖8 TAM抗藥性乳癌細胞株基因模組之基因功能網絡 30
圖9 TAM抗藥性乳癌細胞株基因模組之基因交互作用網絡 30
圖10 TAM抗藥性乳癌細胞株之高度連結性基因網絡 31
圖11 TAM抗藥性乳癌細胞株之關鍵基因路徑 31
圖12 以ROC曲線評估關鍵基因預測臨床乳癌病患對於TAM抗藥性之效度 33
圖13 乳癌細胞對於TAM刺激之細胞存活情形 35
圖14 乳癌細胞在TAM刺激後之關鍵基因表現 36
圖15 乳癌細胞轉染siADM後之ADM基因表現 38
圖16 乳癌細胞轉染siADM後，在TAM刺激下之關鍵基因表現 39
圖18 乳癌細胞對於TAM刺激之細胞凋亡凋亡螢光圖 42
圖19 乳癌細胞對於TAM刺激後細胞凋亡情形之螢光量化圖 43
圖20 乳癌細胞產生抗藥性牽涉之功能及基因表現圖 52

 
表目錄
表1 基因名稱縮寫 VIII
表2 乳癌高危險因子 3
表3 乳癌臨床分期 4
表4 本研究使用之人類乳癌細胞TAM抗藥性相關之基因微陣列數據集 19
表5 本研究使用之人類乳癌細胞之特性 22
表6 本研究所使用之基因引子序列 26
表8 ROC曲線評估關鍵基因預測臨床乳癌病患對於TAM抗藥性效度之數據 34
表9乳癌細胞以TAM刺激後之關鍵基因表現 37
表10 乳癌細胞轉染siADM後，在TAM刺激下之關鍵基因表現 40

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