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研究生:洪玉如
研究生(外文):Yu Ju Hung
論文名稱:微小RNA127促進肺腺癌細胞的侵入能力
論文名稱(外文):MicroRNA127 Promote Lung Adenocarcinoma Cell Invasion
指導教授:蔡孟峯
指導教授(外文):Meng Feng Tsai
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:63
中文關鍵詞:肺癌微小RNA微小RNA127侵入移動目標基因二維電泳
外文關鍵詞:Lung cancermicroRNA(miRNA)miR-127invasionmigrationtarget gene2D-PAGE
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肺癌病患具有高死亡率與高轉移率的特徵。經過治療後,超過五年的存活率仍低於15 %。因此如同其他癌症一般,癌細胞發生轉移為影響肺癌病患存活的關鍵因素。近年來許多微核醣核酸(microRNA, miRNA)被認為可以藉由影響致癌基因、抑癌基因以及調控細胞增生與凋亡(apoptosis)的相關基因參與癌細胞的形成與癌細胞轉移機制。文獻指出,在膀胱癌以及直腸癌的研究中發現miR-127的表現量會降低。然而在子宮頸癌的研究中則顯示,miR-127表現量較高與癌細胞的移動具有正相關性。miRNAs約有21 核苷酸(nucleotide)長,在動物、植物和病毒中都有存在。特定的miRNA可以調控特定基因的功能表現,其所參與的細胞功能包含調控細胞凋亡、胚胎發育、細胞增生(proliferation)和細胞分化等等。因此本研究想要探討miR-127在肺癌中所扮演的角色為何。首先以即時定量聚合酵素連鎖反應(即時定量RT-PCR)檢測正常支氣管上皮細胞(Beas-2B)與肺癌細胞(CL1-0及CL1-5)中miR-127的表現量,結果顯示在肺癌細胞CL1-5中miR-127表現量高於Beas-2B及CL1-0細胞4.9倍。利用pSilencer 3.1 H1 puro 載體的系統在CL1-0細胞中建立miR-127大量表現細胞株(A6、A9、A31、A36)與低表現的對照組(C4、C7、C10)。利用MTT assay與colony formation測試細胞的生長速率,結果顯示miR-127對colony formation不會造成明顯的差異。利用移動與侵入實驗;測試細胞的移動與侵入能力,結果發現在移動能力中,miR-127 A6、A36與mock C10相比較,增加100%的移動能力;在侵入能力中,miR-127 A6、A36與mock C10相比較,增加100 %的侵入能力。而在CL1-5細胞中將miR-127抑制後,實驗結果與CL1-0相同。用預測網址(http://www.targetscan.org/)分析miR-127的目標基因CDX1,也以RT-PCR分析證明,在miR-127大量表現的細胞中(A6、A31及A36),目標基因CDX1會被抑制。另一方面透過二維電泳分析出一個基因:TCP-1,在肺癌細胞中miR-127扮演著促進癌細胞移動與侵入能力的角色。
Lung cancer is the most common cause of cancer death in the world. Non small cell lung carcinoma (NSCLC) is the predominant type of lung cancer. Similar to other malignant tumors, undesirable prognosis for patients with NSCLC has been attributed to the development of metastasis. Therefore, it is important to select and identify the molecular marker indicated patients with lung cancer, especially in terms of invasion and metastatic potential. MicroRNAs (miRNAs) are small noncoding RNAs, assumably 18-24 nucleotides in length that can downregulate various target gene products by translational repression or by directing mRNA degradation. There are many reports showed that miRNAs may be involved in several cellular processes, including metabolism, differentiation, proliferation, cell cycle and apoptosis. In this study, we check miR-127 play role in lung cancer. Real-time quantitative PCR(RT-PCR)identify the using lung cancer cell line model (normal human bronchial epithelial cell: BEAS-2B; lung cancer cell: CL1-0 and CL1-5). Result expression miR-127 in CL1-5 cell high than CL1-0 cell 4.9 folds. Use pSilencer 3.1 H1 puro vector construct high miR-127 expression system in CL1-0 cells(A6, A9, A31 and A36)low expression mock(C4, C7 and C10). Use MTT assay and colony formation analysis cell proliferation. Result expression miR-127 was not effect cell proliferation , to take migration and invasion assay , to discover increased 100% of the migration ability compared with mock C10, in invasion ability, miR-127 A6, A36 increase 100% of the invasion ability compared with mock C10. The result is the same as CL1-0 after inhibiting miR-127 in CL1-5 cells. To use( http://www.targetscan.org/ ) forecast miR-127 target gene CDX1 may with migration, and ayalysis RT-PCR miR-127 high expression cells (A6, A31 and A36) target gene CDX1 will be inhibited. On the other hand analyse a gene through two-dimentional electric swimming: TCP-1. miR-127 promotes the role that moved and invaded ability of cancer cell play in the lung cancer cell.
目錄

封面內頁
簽名頁
授權書 iii
中文摘要 iv
英文摘要 vi
誌謝 viii
目錄 ix
圖目錄 xii

1. 前言 1
1.1 肺癌 1
1.2 癌轉移 3
1.3 癌轉移相關基因研究的發展 5
1.4 微小RNA(Micro RNA, miRNA) 6
1.5 miRNA在細胞的生成與作用 7
1.6 miRNA 生物功能 8
1.7 miRNA與癌轉移 10
2. 研究動機 11
3. 材料與方法 12
3.1 實驗流程 12
3.2 材料與方法 13
3.2.1 即時定量RT-PCR分析 13
3.2.2 細胞株(Human cell lines) 15
3.2.3 DMEM培養液配置 15
3.2.4 細胞繼代培養(Cell subculture) 16
3.2.5 質體DNA萃取 16
3.2.6 構築hsa-miR-127表現載體 17
3.2.6.1 hsa-miR-127片段 17
3.2.6.2 質體與miR-127片段酵素剪切與黏合 17
3.2.6.3 miR-127構築與轉殖大腸桿菌 18
3.2.7 轉染實驗(Transfection) 19
3.2.8 建立提昇hsa-miR-127穩定表現之系統 19
3.2.9 建立抑制hsa-miR-127表現 20
3.2.10 RNA萃取 21
3.2.11 反轉錄聚合酶連鎖反應 (Reverstranscription-
polymerasechain reaction, RT-PCR) 22
3.2.12 細胞增生分析(MTT assay) 23
3.2.13 細胞群落形成分析 (Colony formation assay) 24
3.2.14 細胞基質侵襲力分析(Matrigel invasion assay) 24
3.2.15 細胞移動力分析(Cell migration assay) 25
3.2.16 二維電泳
3.2.16.1 蛋白質製備 26
3.2.16.2 第1維電泳(Isoelectric Focusing) 26
3.2.16.3 第2維電泳(SDS-PAGE) 27
3.2.16.4 銀染 28
4. 結果與討論 30
5. 結論 41
參考文獻 59
附錄 63
參考文獻

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