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研究生:陳健維
研究生(外文):Chien-Wei Chen
論文名稱:微型核糖核酸-378在人類大腸直腸癌中所扮演的角色
論文名稱(外文):The role of miR-378 in human colorectal cancer
指導教授:翁文慧
口試委員:林琦鑫馮思中
口試日期:2012-07-26
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:生物科技研究所
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:2012
畢業學年度:100
語文別:中文
論文頁數:33
中文關鍵詞:大腸直腸癌KRAS/BRAF突變微型核糖核酸-378
外文關鍵詞:colorectal cancerKRAS/BRAF mutationmiR-378
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微型核糖核酸-378是一個大約21鹼基微小片段核糖核酸分子,能夠藉由與信使核糖核酸序列相互結合,導致基因不表達。曾有文獻報導低表達的微型核糖核酸-378 與大腸直腸癌有高度相關。大腸直腸癌多屬於基因突變型癌症,標靶藥物-單株抗體爾必得舒為上皮生長因子受器的阻斷劑用於治療大腸直腸癌已有多年。然而,當腫瘤含有KRAS或BRAF基因突變時,此標靶藥物並不具抑制效果;然KRAS或 BRAF突變型卻分別佔了全大腸直腸癌患者中的40%及10%。微型核糖核酸-378被發現在大腸直腸癌與有絲分裂蛋白激酶訊息傳導途徑的基因中有數個結合位。我們假設微型核糖核酸-378經由轉染作用進入細胞後,也許能夠抑制該途徑中某些致癌蛋白的表現量,進而達到抑制腫瘤細胞增生的效果。在此研究中,我們使用六株經去氧核糖核酸定序法鑑定基因突變型後的人類大腸直腸癌細胞株作為研究模型,利用即時定量反轉錄聚合酶連鎖反應觀察各類型突變細胞之微型核糖核酸-378 轉染前後細胞株中的總表現量,並藉由西方墨點法觀察微型核糖核酸-378轉染前後各相關基因的蛋白表現量。本研究目標在於觀察微型核醣核酸-378 表現量對於 MAPK傳導途徑中的相關蛋白表現量的影響。結果顯示,各致癌蛋白在人類大腸癌細胞株中突變型態不同其表現量也相異,經轉染微型核糖核酸-378 分子後更有明顯蛋白表現量差異,且藥物試驗均呈現有效反應此研究的研究模型提供一重要結論,微型核醣核酸-378藉由調控 MAPK 傳導途徑中促進細胞凋亡以及增生的蛋白表現量,影響細胞的存活率和對於爾必得舒的敏感性。此外,我們推測,ERK蛋白的表現量,在未來可能能夠成為一個指標,去確認轉染入微型核醣核酸-378 後是否可降低細胞的存活率。我們的結果亦發現,微型核醣核酸-378 與爾必得舒合併使用在晚期大腸直腸癌可能比較具有療效。

MicroRNA-378 is a short RNA molecule with 21 base pair that causes gene silencing by the binding of miRNA to their complementary mRNA. Literatures showed low expression of miR-378 correlated with colorectal cancer (CRC). Colorectal cancer is one of the genetic cancers. Cetuximab, a monoclonal antibody, has been used as a target drug for the epidermal growth factor receptor (EGFR) for years. However, when the tumor contain with KRAS or BRAF mutations showed no response to the drug, which account for 40 % and 10 % of CRC patients, respectively. Several binding sites of Mitogen-Activated Protein Kinase (MAPK) pathway were complemented with miR-378 in CRC. We hypothesized transfection of miR-378 into colorectal cancer cell line may suppress the oncoproteins expression of the MAPK pathway. Herein, we using sequencing to identify mutation types of six human colorectal cancer cell line, quantitative RT-PCR to confirmed RNA expression, western blotting to observed oncoproteins expression, then further transfected miR-378 into six CRC cell lines. Our study aimed to observe whether expression level of miR-378 was associated with the oncoproteins in the MAPK pathway. The results showed different level of oncoproteins expression and mutant sites occurred in all cell lines. Transfection of miR-378 reduced the expression level of oncoproteins. The drug test was positive after transfection. This study model provided an important conclusion, the miR-378 can regulate the expression of oncoproteins in MAPK pathway. The influence of cell survival rate and the efficiency of Cetuximab were regulated by miR-378 via suppress protein expression of apoptosis and proliferation in MAPK pathway. The expreesion of ERK protein appears to be a marker for evaluating of miR-378 reduce the cell survival in the future. Our results seem to increase the efficacy of Erbitux after transfected miR-378 in late colorectal cancer cell line.

中文摘要…………………………………………………………………………….....i
英文摘要……………………………………………………………..…………….....iii
誌謝…………………………………………………………….………..………….....v
目錄………………………………………………………………………..……….....vi
表目錄……………………………………………………………………………….viii
圖目錄……………………………………………………………………..………….ix
第一章 緒論…………..………………………………………………….......……...1
1.1大腸直腸癌的病理基因機轉…………………………………………...…...2
1.2微型核糖核酸(micro-RNA)對基因調控的功能與臨床應用價值…….……4
1.2.1生物學特性與機制..…………………………………………………4
1.2.2 微型核糖核酸與癌症腫瘤之相關性…………………….…………5
1.3微型核糖核酸-378在大腸直腸癌症中扮演的角色…….…………..…..…6
第二章 研究目標……………………………………………………………..………9
2.1 研究假設…………………………………….………………………….......9
2.2 研究目標…………………………………….……………………………...9
第三章 實驗方法……………………………………………………………………10
3.1 細胞株……………………………………….……………………………..10
3.2 細胞培養…………………………………….……………………………..10
3.2.1 培養條件…………………………………………………………..10
3.2.2 細胞繼代……………………………………………………………10
3.3 轉染(Transfection) ………………………………………………………...11
3.3.1 實驗原理…………………………………………………………11
3.3.2 實驗流程…………………………………………………………11
3.4 萃取核糖核酸(RNA extraction) …………………………………………..13
3.5 反轉錄(Reverse Transcription) ……………………………………………13
3.5.1實驗原理………………………………………………………….13
3.5.2實驗流程………………………………………………………….14
3.6 即時定量聚合酶連鎖反應(Quantitative Polymerase Chain Reaction)…...16
3.6.1實驗原理………………………………………………………….16
3.6.2實驗流程………………………………………………………….16
3.7 蛋白質萃取(Protein Extraction) …………………………………………..17
3.8標準蛋白曲線繪製與Bradford法蛋白濃度測定…………………………17
3.9西方墨點法(Western Blotting) ……………………………………………..18
3.9.1實驗原理……………………………………………………….....18
3.9.2實驗流程………………………………………………………….18
第四章 實驗結果……………………………………………………………………20
4.1微型核糖核酸-378轉染前後表現量比較…………………………………20
4.2微型核糖核酸-378轉染前後MAPK傳導途徑蛋白質表現量……………21
第五章 討論…………………………………………………………………………26
參考文獻……………………………………………………………………………..30


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