臺灣博碩士論文加值系統

近幾年來，惡性腫瘤持續居十大死因之首，而乳癌為台灣女性第四常見的惡性腫瘤，乳癌在全世界發生率正持續上升。在所有乳癌中，三陰性乳癌約占 15 %，它被定義為缺少雌激素受體、黃體素受體與第二型人類表皮生長因子受體。三陰性乳癌預後極差；另外，典型三陰性乳癌擁有高復發率、高轉移與高死亡率的特性。本研究中使用 2-PN (2-phenylnaphthyridin-4-one) 衍生物藥物 CSC-3436探討對於三陰性乳癌細胞株 MDA-MB-231 的腫瘤生成與基因表現的影響。
實驗結果證實 CSC-3436 可以改變細胞型態、降低細胞存活率、抑制細胞增生、抑制細胞群聚形成能力、抑制細胞非貼附性生長能力與抑制細胞轉移能力。進一步結果表示 CSC-3436 抑制 MMP-2與 MMP-9 之表現。根據 gene expression array 與 microarray 的結果，我們進一步研究並證實 CSC-3436 會透過上調 miR-1290 的表現進而抑制 NAT2 的表現。此外，利用轉染 NAT2 shRNA 實驗，結果證實抑制 NAT2 表現可以抑制細胞增生、細胞非貼附性生長能力與細胞轉移能力並且使細胞型態轉型成類上皮細胞 (epithelial-like)。
綜合以上結果， CSC-3436 可藉由 miR-1290 抑制 NAT2 表現進而抑制人類三陰性乳癌的腫瘤生成能力。因此，CSC-3436可視為一有潛力之抗癌藥物。

Malignant neoplasms have continued to be the top leading cause of death. Recently, breast cancer is the most common malignancy among women, it is the fourth leading cause of cancer death among women in Taiwan, and its incidence is increasing worldwide. Triple-negative breast cancer (TNBC) accounts for approximately 15% of breast cancers, it is defined by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and HER2. Overall, TNBC is characteristically aggressive with poor prognosis, high recurrence, metastatic, and mortality rates. In this study, we used CSC-3436, a derivative of 2-phenylnaphthyridin-4-one (2-PN) to investigate the effects of CSC-3436 on TNBC MDA-MB-231 cell tumorigenesis and gene expression. We demonstrated that CSC-3436 could cause morphological change and suppressed cell viability, proliferation, colony forming ability, anchorage-independent growth and cell migration in MDA-MB-231 cells. Moreover, we showed that CSC-3436 reduced the expression of MMP-2 and MMP-9 in MDA-MB-231 cells. According to these results of gene expression array and microarray, our studies shown that CSC-3436 could down-regulate the expression of N-acetyltransferase 2 (NAT2) via up-regulating the expression of miR-1290. We next used shNAT2 to inhibit the expression of NAT2 and found that shNAT2 stable transfect could cause cell morphology become epithelial-like and suppress cell proliferation, anchorage-independent growth, and cell migration. Finally, our results showed that CSC-3436 could inhibit the expression of NAT2 and repress TNBCs cells tumorigenesis. These findings suggest that CSC-3436 will be a potential anticancer drug for TNBC.
 

考試委員審定書影本 I
中文摘要 II
英文摘要 III
縮寫表 IV
誌謝辭 V
目錄 VII
第一章 前言 1
第一節 研究動機與目的 1
第二章 文獻回顧 2
第一節 癌症 2
附圖一、台灣民國99至100年主要死因死亡人數統計圖 2
附圖二、癌症的六大特性 3
附圖三、新興的癌症特性與進行的特徵 4
第二節 乳癌 (Human breast cancer) 5
附圖四、台灣民國99至100年主要癌症死亡人數占率 5
第三節 三陰性乳癌 (Triple-negative breast cancer；TNBC) 8
第四節 CSC-3436藥物介紹 10
附圖五、化合物結構 (2-PN；2-phenylnaphthyridin-4-ones) 11
附圖六、CSC-3436選擇性毒殺惡性的腫瘤細胞 11
第五節 N-乙醯基轉移酶第二型 (arylamine N-acetyltransferase 2) 的介紹 12
第六節MicroRNAs (miRNAs；miRs)的介紹 14
附圖七、miRNAs的作用機制 16
第三章 研究材料與方法 17
第一節 材料來源 17
第二節 細胞培養 (Cell culture) 22
第三節 細胞型態觀察 (Cell morphology observation) 23
第四節 細胞增生速率分析 (Cell proliferation assay) 24
第五節 細胞存活率分析 (MTT assay) 24
第六節 西方墨點法 (Western blot) 25
第七節 即時定量聚合酶連鎖反應(quantitative real-time polymerase chain reaction；qPCR) 28
第八節 細胞聚落形成能力分析 (Colony formation assay) 35
第九節 軟瓊脂分析 (Soft-agar assay) 35
第十節 Gene expression array與microRNA Microarray analysis 36
第十一節 傷口癒合試驗 (Wound-healing assay) 36
第十二節 細胞轉染 (Transfection) 37
第十三節 明膠蛋白酵素電泳法 (Gelatin-Zymography) 39
第十四節 統計方法 (Statistical Analysis) 41
第四章 研究結果 42
第一節 探討 CSC-3436 對於不同乳癌細胞株之影響 42
第二節 觀察 CSC-3436 對於三陰性乳癌細胞株 MDA-MB-231 腫瘤生成 (tumorigenesis) 能力之影響 43
第三節 觀察 CSC-3436 對於三陰性乳癌細胞株 MDA-MB-231 基因表現 (gene expression) 之影響 (表一) 45
第四節 探討 CSC-3436 與 NAT2 之間的關係 46
第五節 探討 CSC-3436 是否透過抑制 NAT2 之表現進而影響三陰性乳癌細胞株 MDA-MB-231 腫瘤生成 (tumorigenesis) 之能力 48
第六節 探討 CSC-3436 對於三陰性乳癌細胞株 MDA-MB-231 microRNAs 表現之影響 (表二) 54
第五章 討論 60
第六章 結論 64
附圖八、CSC-3436 之作用機制 65
附表一、 SDS-PAGE 膠體製備之配方 66
附表二、 Gelatin-Zymography 膠體配方 67
附表三、 Gelatin-Zymography 之 5X loading dye 配方 68
附表四、 Gelatin-Zymography 之 10X Renature Buffer 配方 68
附表五、 Gelatin-Zymography 之 1X Developing Buffer 配方 68
附表六、 Gelatin-Zymography 之 MMP Staining solution 配方 69
附表七、 Gelatin-Zymography 之Destain solution 配方 69
圖目錄 70
圖一、 CSC-3436 對於不同乳癌細胞存活率 (cell viability) 之影響 70
圖二、 CSC-3436 對於不同類型乳癌細胞非貼附性生長之影響 71
圖三、 CSC-3436對 MDA-MB-231 細胞型態之影響 72
圖四、 CSC-3436 對於 MDA-MB-231 增生速率之影響 73
圖五、 CSC-3436 對於 MDA-MB-231 接觸性抑制生長能力之影響 74
圖六、 CSC-3436 對於 NAT2 mRNA 表現之影響 75
圖七、 CSC-3436 對於 NAT2 蛋白質表現之影響 76
圖八、 CSC-3436 對於 NAT1 蛋白質表現之影響 77
圖九、探討 CSC-3436 處理的時間長短是否會影響 NAT2 mRNA之表現 78
圖十、 不同 stable clone 抑制 (knock-down) NAT2 之效率 79
圖十一、 shNAT stable clones 之細胞型態 80
圖十二、 抑制 NAT2 表現對於 MDA-MB-231 細胞增生速率之影響 81
圖十三、 抑制 NAT2 表現對於 MDA-MB-231 細胞非貼附性生長能力之影響 82
圖十四、與圖十五、 探討 CSC-3436 對於 MDA-MB-231 細胞轉移能力之影響 83
圖十六、 CSC-3436 對於 MMP-2 與 MMP-9 活性之影響 84
圖十七、 探討 CSC-3436 對於 MDA-MB-231 細胞爬行 (migration) 能力之影響 85
圖十八、 抑制 NAT2 表現對於 MMP-2 與 MMP-9 蛋白質表現之影響 86
圖十九、抑制 NAT2 表現對於MDA-MB-231 細胞爬行 (migration) 能力之影響 87
圖二十、 miR-1290 與 NAT2 互補之序列圖 88
圖二十一、 CSC-3436對 miR-1290 表現之影響 89
圖二十二、 CSC-3436 與 miR-1290 抑制劑對於 miR-1290 表現量之影響 90
圖二十三、 CSC-3436 與 miR-1290 抑制劑對於 NAT2 mRNA 表現量之影響 91
圖二十四、CSC-3436 與 miR-1290 抑制劑對於 NAT2 蛋白質表現量之影響 92
圖二十五、 CSC-3436 與 miR-1290 抑制劑對於 MMP-2 與 MMP-9 蛋白質表現量之影響 93
圖二十六、 CSC-3436 與 miR-1290 抑制劑對於 MDA-MB-231 細胞爬行能力之影響 94
表目錄 95
表一、 Gene expression array 分析CSC-3436對MDA-MB-231 細胞基因表現 95
表二、 CSC-3436 對於三陰性乳癌細胞株 MDA-MB-231 microRNAs 表現之影響 96
第七章 參考文獻 97

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