臺灣博碩士論文加值系統

O-GlcNAcylaion是一種蛋白質轉譯後修飾，其修飾是將單一的N-acetylglucosamine(GlcNAc)添加到細胞核與細胞質蛋白上的絲氨酸或蘇胺酸，此種修飾參與調控代謝、基因轉錄以及轉譯，且具有可逆、動態、可誘導的特性，分別由兩個酵素進行催化： O-GlcNAc transferase (OGT)負責添加以及O-GlcNAcase (OGA)負責移除GlcNAc修飾。在肺癌中，OGT的mRNA以及蛋白質在腫瘤當中表現量比正常的組織要來的高，且較高的OGT表現和病人較差的存活率有關聯。微核醣核酸(miRNA)在基因表現的調控中扮演重要角色，可透過結合到3’UTR調節mRNA的穩定性或轉譯的效率，已知miRNA的異常調控和腫瘤的進程有關。然而，在肺癌細胞中OGT的表現量是如何被調控並不清楚。在本篇研究中，我們致力於在肺腺癌細胞中尋找是否有miRNAs會結合到OGT的3’UTR上。利用螢光素酶分析對OGT的3’UTR分析，我們發現在OGT的3’UTR +3881到+5482這段序列有可能參與抑制OGT的表現，利用四種不同運算方法的miRNA-mRNA網站作預測，發現16個可能結合到OGT之3’UTR上的miRNAs。在這16個miRNA當中，我們發現miR-200a-3p對於帶有OGT-3’UTR之報導基因的螢光素酶活性具有最明顯的抑制效果。我們也利用點突變實驗確認miR-200a-3p可能會直接透過OGT的3’UTR去OGT表現。除此之外，過度表現miR-200a-3p會去抑制OGT蛋白的表現。

O-GlcNAcylation is a form of post-translational protein modification in which a single N-acetylglucosamine(GlcNAc) is added to serine or threonine residues of nuclear and cytoplasmic proteins. This modificationis involved in regulating metabolism, gene transcription and translation. O-GlcNAcylation is a reversible, dynamic and inducible modification that is catalyzed by two enzymes: O-GlcNAc transferase (OGT) is responsible for the addition and O-GlcNAcase (OGA) mediates the removal of GlcNAc from proteins respectively. In lung cancer, the expression of OGT mRNA and protein is higher in tumor tissues than that in normal tissues, and the high expression of OGT is associated with poorer survival of patients. MicroRNAs (miRNAs) plays an important role in modulating gene expression by regulating mRNA stability or translation efficiency through binding to 3’UTR, and dysregulation of miRNAs is involved in tumor progression. However, the underlying mechanism that regulates OGT expression in lung cancer cells is still unclear. Therefore, we aimed to explore if the OGT gene can be targeted by any miRNAs through its 3’UTR in lung adenocarcinoma cells. Our analysis of the 3’UTR of OGT by luciferase reporter assays revealed that the+3881 to +5482 region in OGT-3’UTR may participate in repressing OGT expression. By using four different algorithms to predict miRNA-mRNA interaction, sixteen miRNAs that may target OGT-3’UTR were obtained. Among these sixteen candidates, we demonstrated that miR-200a-3p could most significantly decrease the luciferase activity expressed from a reporter gene carrying OGT-3’UTR. We also used site-directed mutagenesis to confirm that miR-200a-3p may directly regulateOGT expression through OGT-3’UTR. Moreover, OGT protein levels were repressed upon overexpressing miR-200a-3p.

致謝……………………………………………………………………………….…i
Abstract………………………………………………………………………….…ii
摘要………………………………………………………………………………....iii
目次………………………………………………………………………………....iv
第一章、 緒論……………………………………………………………….…….1
1.1肺癌………………………………………………………………………….….1
1.2 O-GlcNAc Transferase (OGT)與O-GlcNAcylation………………………..1
1.3 O-GlcNAc Transferase (OGT)與癌症……………………………………….2
1.4 MicroRNA(s) 與O-GlcNAc Transferase (OGT)…………………………...3
第二章、 實驗材料與方法………………………………………………….…….6
2.1 材料…………………………………………………………………….……....6
2.1.1細胞株 (Cell line) …………………………………………………..............6
2.1.2勝任細胞 (Competent cell) ……….………………………………….…....6
2.1.3引子 (Primer) ………………………………………………………….…....6
2.1.4抗體 (Antibody).…………………….……….….………...……….….…....6
2.1.5緩衝溶液 (Buffer) ………………….…….…………….………….…..…....6
2.1.6質體 (Plasmid) …..…….………………....………………..………….…....6
2.1.7酵素、Kit與其他試劑………………………………………………….…....7
2.2 方法……………………………………………………………………….…....8
2.2.1細胞培養 (Cell culture) ……………………………………………….…....8
2.2.2質體建構 (Plasmid construction) ……………………………………........8
2.2.3細胞轉染 (Cell transfection).….………………………..………….…......10
2.2.4微核糖核酸表現量偵測 (miRNA expression detection ) ………....…....10
2.2.5細胞株蛋白質表現測量 (Protein expression detection).……………....12
2.2.6報導基因試驗 (Reporter assay) ………......................................…....13
2.2.7統計分析...……...…………………………………………………….........14
第三章、 結果……...……...……………………………………………….........15
3.1 OGT-3’UTR(+3881-+5482)可能參與調控OGT表現…..........................15
3.2利用miRNA資料庫預測可能結合在OGT-3’UTR上的miRNA(s)........15
3.3研究miRNA(s)調控OGT-3’UTR能力之分析…......................................16
3.4 過量表現miRNA-200a-3p抑制OGT蛋白表現….................................17
3.5抑制miRNA-200a-3p表現會促進luciferase activity…………………..…18
第四章、 討論….........................................................................................19
第五章、 參考文獻......................................................................................22
第六章、 圖…..............................................................................................25
第七章、 表…..............................................................................................32
附錄……………………………………………………………………………….38

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