臺灣博碩士論文加值系統

惡性腫瘤在台灣一直是十大死因之首，乳癌是女性死亡率排名第四。造成惡性腫瘤形成的原因之一為基因失調，基因失調包含致癌基因的過度表現、抑癌基因的失活以及表觀遺傳學的改變包含為小核醣核酸(microRNA)表現差異。MicroRNA負調控基因且參與許多細胞功能包括細胞凋亡、代謝、增殖與腫瘤轉移等。經過定量反轉錄聚合酶鏈鎖反應檢測，發現在高轉移的三陰性乳癌細胞MDA-MB-231中，miR-194表現量較低，文獻指出 miR-194在不同癌症中可作為致癌基因亦可為抑癌基因，但是在乳癌中並無被報導過。因此，本研究主要探討miR-194在乳癌轉移中扮演之角色並探討其調控機制。結果顯示，藉由細胞傷口癒合和細胞爬行實驗發現轉染miR-194後的MDA-MB-231的爬行能力下降。根據明膠酵素實驗中，基質金屬蛋白酶MMP-9及MMP-2的活性下降。此外使用西方墨點法分析，發現轉染miR-194會使轉移相關的蛋白表現量下降且會讓黏附相關蛋白表現量上升。接著使用TargetScan網站找到miR-194調控的標靶基因，例如AKT2、Bmi1、SOX5和FOXA1等，並使用Western blotting確認該標靶基因的表現量下降。本研究結果顯示miR-194具有抑制乳癌細胞轉移的功能。

Malignant carcinoma has been the leading cause of disease death in Taiwan. The mortality of breast cancer ranks fourth in female. Carcinogenesis is associated with oncogene activation, tumor suppressor gene inactivation and epigenetic changes including difference in microRNA expression. MicroRNAs negatively regulate gene expression and are involved in several cell functions including apoptosis, metabolism, proliferation and metastasis. Relative expression of miR-194 is lower in metastatic triple-negative breast cancer cell line (MDA-MB-231) than in non-metastatic triple-negative breast cancer cell line (MCF7). Several lines of evidence showed that miR-194 can be oncogene or tumor suppressor gene in different cancers. However, there is no report about the role of miR-194 in breast cancer. Therefore, this study was to investigate the biological functions of miR-194 in breast cancer carcinogenesis. The results revealed that migration of MDA-MB-231 was inhibited by miR-194 using wound healing and transwell assays. According to the zymography data, miR-194 inhibited the gelatin-degrading activity of MMP-9 and MMP-2. In addition, miR-194 increased the expression of epithelial marker and decreased that of mesenchymal marker by western blotting. To verify miR-194 target genes, the TargetScan program is used to search miR-194 target genes including AKT2, Bmi1, FOXA1 and SOX5. The protein expression levels of miR-194 target genes were decreased after transfecting the miR-194 precursor into breast cancer cells. This study proves that miR-194 can inhibit EMT for breast cancer.

中文摘要 I
Abstract II
總目錄 III
圖目錄 V
符號與縮寫 VI
第一章、緒論 1
第一節 癌症 1
第二節 乳癌 1
第三節 三陰性乳癌 (Triple negative breast cancer, TNBC) 2
第四節 癌症的侵襲與轉移 2
第五節 MicroRNA 3
第六節 MicroRNA和上皮與間質轉移的關係 4
第七節 miR-194與其標靶基因 4
第二章、研究動機與目的 5
第三章、結果與討論 6
第一節 結果 6
一、miR-194的表現量在MDA-MB-231中較低 6
二、miR-194對MDA-MB-231之細胞爬行影響 6
三、miR-194對MDA-MB-231之MMP-2及MMP-9活性影響 7
四、miR-194對MDA-MB-231腫瘤轉移相關蛋白表現量影響 7
五、使用冷光報導基因實驗確認miR-194的標靶基因 7
六、使用西方墨點法檢測miR-194的標靶蛋白 8
第二節 討論 9
第四章、結論 11
第五章、實驗部分 12
第一節 實驗材料 12
一、細胞株與MicroRNA 12
二、藥品與試劑 12
三、儀器設備 16
第二節 實驗方法 17
一、細胞解凍 17
二、細胞繼代與培養 17
三、細胞冷凍與保存 19
四、萃取RNA 20
五、定量反轉錄聚合酶反應 21
六、建構標靶基因之冷光報導質體 22
七、萃取質體 DNA 及定量 23
八、轉染質體 DNA 至細胞 24
九、冷光報導基因實驗 (Luciferase reporter assay) 24
十、細胞傷口癒合實驗 (Wound-healing assay) 25
十一、細胞遷移實驗 (Cell migration assay) 26
十二、明膠酵素實驗 (Gelatin Zymography assay) 26
十三、細胞蛋白萃取與定量 30
十四、西方墨點法 (Western Blotting) 31
十五、統計方法 34
第六章、圖表 35
第七章、附錄圖 48
第八章、參考文獻 54

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