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系統識別號 U0026-1808201510310700
論文名稱(中文) TGIF誘發之Hedgehog 路徑活化參與在有gemcitabine抗藥性的尿路上皮癌
論文名稱(英文) TG-interacting factor-induced Hedgehog pathway activation is involved in the gemcitabine resistance of urothelial carcinoma
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 103
學期 2
出版年 104
研究生(中文) 黃悅筑
研究生(英文) Yue-Jue Huang
學號 T36021023
學位類別 碩士
語文別 英文
論文頁數 52頁
口試委員 指導教授-黃暉升
召集委員-蔡宗欣
口試委員-洪良宜
口試委員-張權發
中文關鍵字 尿路上皮癌  gemcitabine 抗藥性  Hedgehog 路徑  AKT  GSK3β 
英文關鍵字 UC  gemcitabine resistance  Hedgehog pathway  AKT  GSK3β 
學科別分類
中文摘要 在台灣,尿路上皮細胞癌 (Urothelial carcinoma, UC) 是泌尿道系統中最常發生的癌症之一。尿路上皮細胞癌的治療方式是手術搭配化療,目前 gemcitabine 和 cisplatin 兩種藥物的合併使用已被應用作為標準化療方案。但是尿路上皮細胞癌的化療常常面對高復發性和抗藥性的問題,其中 gemcitabine 出現抗藥性常常是治療失敗的主要原因之一,所以釐清抗藥性機轉對於治療會有很大的幫助。近年來,越來越多的研究顯示 Hedgehog 路徑在癌症發展及抗藥性中扮演重要的角色。之前我們的研究發現 TGIF 在尿路上皮細胞癌癌化過程中會增加細胞遷移/侵犯的能力,並且可以經由 PI3K/AKT 的通路造成 gemcitabine 抗藥性。而在 Hedgehog 路徑中, GSK3β 在調控 Hedgehog 路徑的活化扮演重要的角色,並且 GSK3β 可作為 PI3K/AKT 通路的一個下游因子。故此篇研究的目的主要是想探討 TGIF 和 Hedgehog 路徑在 gemcitabine 造成的抗藥性中所扮演的角色。起初,我們比對原始尿路上皮癌細胞株 NTUB1 細胞和具有對 gemcitabine 的抗藥性尿路上皮癌細胞株 NTUB1 (NG1.5),結果發現在 NG1.5 細胞株中,TGIF、AKT、GSK3β 和 Hedgehog 因子是過度表現。並且過度表現其中一個 Hedgehog 因子 gli2 會減弱 NTUB1 細胞對gemcitabine 的敏感性而降低 gli2 會增加 NGR 細胞對 gemcitabine 的敏感性。接著,我們在 NTUB1 細胞中利用轉染技術分別將 TGIF 和 AKT1 過度表現還有以gemcitabine 治療 NTUB1 細胞,結果顯示在 GLI 熒光素酶報告基因 (GLI-luciferase reporter) 中 gemcitabine、TGIF 及 AKT1 都會促使 Hedgehog 路徑活化,gemcitabine 會導致 TGIF、P-AKT 及 P-GSK3β 表現增加,TGIF 會導致 P-AKT 及 P-GSK3β 表現增加,AKT1 會導致 P-GSK3β 表現增加。另外,我們使用 GSKS9D 可抑制 GSK3β 活性並促使 Hedgehog 路徑活化。相反地, 我們使用 GSKS9A 可增加 GSK3β 活性並抑制 Hedgehog 路徑活化。因此,由上述的初步結果來看,我們認為 gemcitabine 會促使 TGIF 表現增加並藉由AKT/ GSK3β 通路促使 Hedgehog 路徑活化最後導致抗藥性生成。然而,還是需要再更進一步的實驗去清釐尿路上皮癌中 gemcitabine 抗藥性的分子機轉。
英文摘要 Urothelial carcinoma (UC) is one of the most common genitourinary tract malignancies in Taiwan. Treatment options for people with UC are chemotherapy with gemcitabine/cisplatin after surgery. However, the chemotherapy of UC usually faced the problem with high recurrence and drug-resistance. Gemcitabine-resistance is one of the important reasons about failure of therapy, and extensive research has been done to elucidate the drug-resistant mechanism. A growing of facts that aberrant Hedgehog pathway has been linked to many cancers and drug-resistance. Previously, we have demonstrated that TG-interacting factor (TGIF) is associated with migration/invasion and poor prognosis in UC. In addition, TGIF also contributed to gemcitabine-resistance in UC via PI3K/AKT pathway. In Hedgehog pathway, glycogen synthase kinase 3β (GSK3β) was considered to play an important role in regulating Hedgehog pathway. In gemcitabine-resistant NTUB1 (NG1.5) cells, increases of TGIF, pAKTser473, pGSK3βser9 and Hedgehog components were found. Overexpression of gli2 increase resistance to gemcitabine in NTUB1 cells, whereas gli2 knockdown reverse resistance to gemcitabine in NG1.5 cells. Treatment with gemcitabine, overexpression of TGIF or AKT1 in NTUB1 cells also increased activity of GLI-luciferase reporter. Therefore, TGIF might activate Hedgehog pathway via AKT/GSK3β signaling to induce resistance of gemcitabine was hypothesized. Overexpression of TGIF increased pAKTser473 to induce Hedgehog pathway. Overexpression of AKT1 could phosphorylate pGSK3βser9 to inactivate its kinase activity and induce Hedgehog pathway. In addition, overexpression of GSKS9D could inactivate GSK3β kinase and increase activity of Hedgehog pathway, however, overexpression of GSKS9A could activate GSK3β kinase to supress Hedgehog pathway. Taken together, these results suggest that gemcitabine can increase TGIF expression to induce AKT/GSK3β signaling to activate Hedgehog pathway, which results in the resistance of UC. Further studies will be required to completely understand the molecular mechanisms involved in gemcitabine resistance of UC.
論文目次 List of Contents
中文摘要…………………………………………………………….……………Ⅰ
ABSTRACT……………………………………………………….………….….Ⅲ
誌謝……………………………………………………...…………………….…Ⅴ
List of Contents……………………………………….………………………….Ⅵ
List of Figures…………………….………………………………………..…….Ⅶ
List of Abbreviations…………….………………………………………………Ⅷ
INTRODUCTION……………….………………………………………..…..…1
HYPOTHESIS…………………………………………………..………………….8
MATERIALS AND METHODS…………………………………………………..9
RESULTS………………………………………………………...…….…….……20
DISCUSSION…………………………………………………………….….……24
REFERENCES……………………………………………………………………28
FIGURES……………………………………………………………….…………33
APPENDIX…………………………………………………………..……………50
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