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研究生:馮彥博
研究生(外文):Yen-Po Feng
論文名稱:O-GlcNAc Transferase在腫瘤細胞DNA受損的表現及調控
論文名稱(外文):Expression and regulation of O-GlcNAc transferase in tumor cells responding to DNA damage
指導教授:周德盈
指導教授(外文):Teh-Ying Chou
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:103
語文別:中文
論文頁數:49
外文關鍵詞:O-GlcNAc transferaseCisplatinOGTDNA damage
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O-GlcNAcylation為一種蛋白質轉譯後修飾,廣泛影響細胞的各種功能;細胞內O-GlcNAc修飾異常可能導致疾病或與癌症發生有關。目前已知調控細胞內O-GlcNAc程度的主要酵素為O-GlcNAc transferase (OGT) 及O-GlcNAcase (OGA)。OGT蛋白將單醣N-acetylglucosamine轉移到蛋白的絲胺酸 (Ser) 或蘇胺酸 (Thr),形成O-linked醣基化;OGA蛋白則可移除此種修飾。OGT蛋白與乳癌細胞的抗藥性有密切關係。利用shRNA將OGT表現抑制後,乳癌細胞MCF7對於Tamoxifen更加敏感。但對於OGT蛋白在癌細胞抗藥性的確切機制,目前還未完全了解。我們利用UV、Adriamycin、和Cisplatin三種處理方式,使肺癌細胞走向死亡的情況下,發現細胞內OGT的RNA及蛋白表現量均會減少。我們也同時找到會被負調控的OGT上游最短啟動子片段,藉由生物資訊庫搜尋,找到可能會結合於這一段啟動子片段的轉錄因子: p53、C/EBP β、Sp1、E2F和GATA-1蛋白等。我們發現無論細胞內具有野生型、突變型p53或無p53蛋白表現,經UV和Adriamycin處理後,OGT蛋白量皆明顯下降,甚至以外送方式大量表現p53也不會影響OGT的蛋白及RNA表現。C/EBP β大量表現在肺癌細胞中,也同樣不會影響OGT蛋白及RNA表現。Sp1蛋白大量表現後,OGT蛋白表現明顯上升,但是RNA表現沒有差異。直到目前為止,我們已初步排除p53、C/EBP β和Sp1蛋白調控OGT上游啟動子的可能性,將繼續尋找調控OGT表現的轉錄因子。另外,我們嘗試探討OGT蛋白在細胞抗藥性的角色時,以外送方式大量表現OGT並無法挽回經過Cisplatin處理後細胞內OGT的蛋白表現量,表示細胞調控OGT蛋白表現機制遠比我們想像複雜許多。
O-GlcNAcylation, one of the post-translational modifications of proteins, participates in diverse cellular mechanisms. Abnormal cellular O-GlcNAc levels may result in diseases such as diabetes and cancer. It is known that O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) play important roles in maintaining cellular O-GlcNAcylation homeostasis. OGT catalyzes O-linked glycosylation of proteins by transferring the monosaccharide, N-acetylglucosamine, onto hydroxyl groups of serine/threonine residues, while OGA reverses this modification. OGT has been shown to be associated with chemotherapy resistance in breast cancer cells; cells with OGT deficiency are more sensitive to Tamoxifen-induced cell death. However, the exact mechanism of OGT-mediated chemotherapy resistance remains unclear. We found that the RNA and protein levels of OGT were decreased in tumor cells under the treatment of UV, adriamycin or cisplatin. We mapped the shortest region in the OGT promoter responding to cisplatin treatment by promoter-reporter assays and used bioinformatics database to predict possible transcription factors that may bind to this region; the candidates we found included p53, C/EBP β, Sp1, E2F and GATA-1. We used cell lines expressing either wild-type p53, mutated p53 or no p53 to observe OGT expression under UV and adriamycin treatment. The results showed that OGT protein levels were decreased under UV and adriamycin treatment regardless of cellular p53 status. Furthermore, overexpression of p53 did not change the protein and RNA levels of OGT. C/EBP β did not have effects on OGT expression, either. Sp1 overexpressed in tumor cells caused an increase in OGT protein levels, but not in RNA levels. Therefore, we have excluded the possibility of p53, C/EBP β, and Sp1 being the transcription factors regulating OGT expression. Moreover, we have tried to study the possible role of OGT in chemotherapy resistance but exogenous expression vectors were not able to restore the expression of OGT under cisplatin treatment. These results suggest that the regulation of OGT in tumor cells is far more complicated than we ever imagined.
中文摘要 6
英文摘要 7
第一章、緒論 8
一、癌症 (Cancer) 8
二、癌症與抗藥性 (Cancer and drug resistance) 8
2-1. 癌症的抗藥性 8
2-2. 抗藥性機制 9
2-3. 目前克服抗藥性的研究 9
三、蛋白質O-link醣基化修飾 (O-GlcNAcylation) 10
3-1. O-GlcNAcylation介紹 10
3-2. O-GlcNAcylaion 於細胞中扮演的角色 10
3-3. O-GlcNAcylation與疾病的關係 11
四、O-linked醣基轉移酶 OGT (O-GlcNAc transferase) 12
4-1. OGT蛋白 12
4-2. OGT蛋白與疾病的關係 12
五、研究動機 (Research motive) 13
第二章、實驗材料與方法 14
一、實驗材料 14
1. 細胞株 (Cell line) 14
2. 勝任細胞 (Competent cell) 14
3. 抗體 (Antibody) 14
4. 引子 (Primer) 14
5. 質體 (Plasmid) 15
6. 實驗溶液 (Buffer) 15
7. 酵素 (Enzyme) 及Kits 16
8. 其他物品 17
二、實驗方法 17
1. 細胞培養 (Cell culture) 17
2. 細菌培養 (Bacterial culture) 18
3. 質體(Plasmid)抽取與檢驗 18
4. 細菌轉型作用 (Transformation) 18
5. 細胞轉染 (Cell transfection) 18
6. 細胞株RNA測量 19
7. 細胞株蛋白質表現測量 20
8. 冷光活性方法 (Luciferase assay) 21
9. 細胞死亡群測量 (Flow cytometry) 22
10. 統計分析 22
第三章、結果 23
第四章、討論 28
第五章、圖 31
圖一、 腫瘤細胞經紫外光及臨床使用的化療藥物等處理後,細胞死亡族群 (Sub G1 population) 增加 31
圖二、OGT蛋白在經紫外光處理的腫瘤細胞內表現量明顯下降 32
圖三、OGT蛋白在經Adriamycin處理的腫瘤細胞內表現量明顯下降 33
圖四、OGT蛋白表現隨著CDDP處理腫瘤細胞時間增加而逐漸下降 34
圖五、OGT的RNA表現隨著CDDP處理腫瘤細胞時間增加而逐漸下降 35
圖六、在經CDDP處理的A549細胞內OGT上游啟動子活性下降的最短長度為500 bps 36
圖七、經由生物資訊找到C/EBP β、E2F、GATA-1、p53、Sp1等預期會調控OGT表現的轉錄因子 37
圖八、OGT蛋白在經紫外光處理的CL1-5和H1299細胞內表現量明顯下降 38
圖九、OGT蛋白在經Adriamycin處理的CL1-5和H1299細胞內表現量明顯下降 39
圖十、在H1299細胞內大量表現野生型 p53蛋白,不會影響OGT蛋白及RNA表現量 40
圖十一、在H1299細胞內大量表現C/EBP β蛋白,不會影響OGT蛋白及RNA表現量 41
圖十二、Sp1蛋白在經CDDP處理的A549細胞內表現量明顯下降 42
圖十三、在H1299細胞內大量表現Sp1蛋白,OGT蛋白表現量明顯上升,RNA表現量則無明顯差異 43
圖十四、由於以大量表現OGT蛋白方式無法挽救CDDP造成細胞內OGT表現減少,因此細胞死亡族群並沒有明顯差異 44
第六章、參考文獻 45

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