臺灣博碩士論文加值系統

MicroRNA (miRNA)是一種進化過程中高度保守的，內源性非編碼的小RNA，它的功能就像是基因表現的負調節器。miRNA可以廣泛的調控生物功能，例如:細胞增生、分化或是細胞凋亡。近來有研究顯示，miRNA的突變或異常表現與多種人類癌症有密切關係，同時也指出miRNA的功能可以像是腫瘤抑制基因或是致癌基因。在我們的研究當中，藉由微陣列分析技術來觀察口腔癌細胞株與正常人類口腔角質細胞的miRNA表現圖譜，找尋與口腔鱗狀上皮細胞癌相關的標的基因。在微陣列分析的結果，我們發現在正常人類口腔角質細胞當中有一些miRNAs (miR185、miR320、及miR340)表現量較口腔癌細胞株高。藉由電腦程式分析來預測這些miRNAs所調控的標的基因，結果偵測出β-catenin有可能為這三種miRNAs共同的標的基因。我們進一步的分析這些miRNAs是否能在後轉錄階段去影響β-catenin的表現，利用特定miRNA的前驅分子或特定miRNA抑制劑我們證實miR185、miR320、及miR340都能影響β-catenin蛋白質的表現量而不影響其RNA的量，而且也證實miR185、miR320、及miR340能透過結合β-catenin 3’UTR而調控β-catenin的表現，並參與在Wnt signaling pathway調節生物功能。這些結果可以更深入的幫助瞭解口腔鱗狀上皮細胞癌形成的分子機轉，同時找尋出新的口腔癌治療與診斷的分子生物標記。

MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators. It could control a wide range of biological functions such as cellular proliferation, differentiation and apoptosis. Recent evidence has shown that miRNA mutations or mis-expression were correlated with various human cancers and indicated that miRNAs can function as tumor suppressors and oncogenes. In this study, we performed the miRNAs expression profiles of oral cancer cells (OC2) and normal human oral keratinocytes (NHOK) by microarray analysis. We identified several miRNA genes that were significantly differently expressed between these two cell lines. To find the possible target genes of miRNAs by using the bioinformatic tools at the miRBase target database, β-catenin was predicted to be a target of three down-regulated miRNAs (miR185, miR320 and miR340) in oral cancer cells. Computational analysis using miRanda software was also identified β-catenin as a target of these miRNAs. Furthermore, we found that expression of β-catenin is negatively regulated by these three miRNAs at a posttranscriptional level. These results may provide the useful information for the exploring of molecular mechanisms in OSCC progression and might also be powerful targets for diagnosis or treatment of OSCC in the future.

中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
表目錄 VIII
圖目錄 IX
緒 論 1
研究動機 8
材料與方法 9
一、正常人類口腔角質細胞(NHOK)初代培養 9
1-1 正常人類口腔角質細胞(NHOK)初代培養 9
1-2 正常人類口腔角質細胞繼代培養 10
1-3 細胞保存 10
1-4 細胞計數 11
二、細胞RNA的萃取 12
2-1 總RNA萃取 12
2-2 大片段與小片段RNA之萃取 12
2-3 RNA濃度定量 14
2-4 反轉錄聚合酵素連鎖反應 14
2-5 聚合酵素連鎖反應 15
2-6 膠體電泳 17
2-7 DNA濃度定量 17
三、miRNA基因微陣列MiRNA Microarray 18
3-1 小片段RNA標記Smalll RNA Labeling 18
3-2 晶片前處理 19
3-3 前雜交反應(Pre- hybridization) 20
3-4 雜交反應(Hybridization) 20
3-5 資料影像掃描與分析 21
3-6 MiRNA目標基因預測 21
3-7 即時定量聚合酵素連鎖反應 21
四、PMIR- REPORT β-catenin 3’UTR expression vector質體構築 23
4-1 DNA片段的選殖(Cloning) 23
4-2 DNA片段之純化 24
4-3 DNA 5’端去磷酸化(Dephosporylation) 24
4-4 接合作用(Ligation) 25
4-5 轉型作用(Transformation) 25
4-6 小量質體萃取(Small-scale preparation of plasmid) 26
五、 報導基因表現分析Luciferase Reporter Assay 27
5-1 MicroRNA轉染與報告基因轉染 27
5-2 Luciferase與β-galactosidase訊號偵測 28
六、MiRNA與目標基因作用之試驗 29
6-1 MiRNA轉染MiRNA transfection 29
6-2 細胞蛋白樣本收集 30
6-3 SDS-PAGE蛋白質電泳 30
6-4 西方點墨法(Western blotting) 32
七、細胞增生分析(Cell prolifrration assay) 34
結 果 35
討 論 40
參考文獻 48
附錄一：儀器 68
附錄二：英文縮寫 70
自述 71

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