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博碩士論文 etd-0912116-092325 詳細資訊
Title page for etd-0912116-092325
論文名稱
Title
探討TMCO1基因在人類膀胱癌細胞株調控AKT去磷酸化之機制
Studies on the mechanism of the TMCO1 dephosphorylates AKT in human bladder cancer cell lines
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
42
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2016-10-11
繳交日期
Date of Submission
2016-11-05
關鍵字
Keywords
AKT去磷酸酶、細胞週期停滯、膀胱泌尿路上皮癌、AKT激酶活性、Transmembrane and coiled-coil domains 1 (TMCO1)
Urinary bladder urothelial carcinoma (UBUC), Cell cycle arrest, AKT phosphatase, AKT kinase activity, Transmembrane and coiled-coil domains 1 (TMCO1)
統計
Statistics
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中文摘要
儘管有效管理方略,膀胱泌尿路上皮癌的復發與進一步發展較惡性癌症機率仍高且對於發展為更惡性膀胱癌患者預後不佳。無疑地,須深入探討癌症進展的分子機制與發展出新穎治療方針。除了在細胞增生、細胞遷移扮演重要角色,Serine/threonine protein kinase 1 (AKT1)亦透過調控週期素依賴激酶抑制劑(Cyclin-dependent kinases inhibitors; CKIs) p21Cip1與p27Kip1的活性,進而促進細胞存活與細胞週期進行。Transmembrane and coiled-coil domains 1 (TMCO1) 在生物界具高度保留性且在成人組織中均有其基因表現,然而在膀胱癌所扮演角色仍未釐清。此研究旨在探討TMCO1如何調控AKT1 (S473)去磷酸化。研究顯示,無論TMCO1過度表現或抑制其表現,PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1) 與PHLPP2蛋白質表現未受其調控。由免疫沉澱結果推測TMCO1蛋白質吸引PHLPP2且兩者在膀胱癌細胞的細胞質共同區域化現象(Colocalization)。除此之外,在細胞試驗中抑制TMCO1表現使AKT1磷酸化增加,進而促使下游p21Cip1 (T154)與 p27Kip1 (T157)磷酸化,以防止CKIs所引起細胞週期停滯現象。總結,TMCO1在人類膀胱癌細胞中具有抑制腫瘤細胞能力,可作為膀胱癌的生物指標或更甚者應用於膀胱癌治療標的。
Abstract
Despite the available management strategies, the recurrence and progression rates of urinary bladder urothelial carcinoma (UBUC) are still high and therapeutic outcomes for patients with advanced bladder carcinoma remain unsatisfactory. It is undoubtedly requires more investigations into the molecular mechanisms of tumorigenesis and the development of novel targeted therapeutics. The AKT1 (Serine/threonine protein kinase 1) plays an important role during cell proliferation, migration and modulates activity of cyclin-dependent kinases inhibitors (CKIs), which includes p21Cip1 and p27Kip1, to promote cell survival and cell cycle progression. Transmembrane and coiled-coil domains 1 (TMCO1) is a highly conserved gene expressed in adult human tissue. However, the function of TMCO1 in bladder cancer remain poorly understood. This study aimed to explore how TMCO1-mediated dephosphorylation of AKT1 at S473. The results shown either gene overexpression or knockdown, TMCO1 did not modulate PHLPP1 and PHLPP2 protein level. It suggested that TMCO1 protein recruited PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2) by immunoprecipitating and colocalication of both in the cytoplasm of UBUC-derived cells. In addition, TMCO1 silencing increased the phosphorylation of AKT1, which could phosphorylate both p21Cip1 on T154 and p27Kip1 on T157, caused retention of CKIs in the cytoplasm and thus, prevention of cell cycle arrest induced by p21Cip1 and p27Kip1 in vitro. It is suggested that potential tumor cell suppressor role of TMCO1 in human bladder cancer and it may serve as biomarker or even a therapeutic target of bladder cancer.
目次 Table of Contents
論文審定書 i
致謝 ii
摘要 iii
Abstract iv
目錄 v
圖次 vii
表次 viii
英文縮寫表 ix
壹、 緒論(Introduction) 1
一、 膀胱癌 1
二、 Transmembrane and coiled-coil domains 1 (TMCO1) 2
三、 AKT與細胞週期 2
貳、 材料與方法(Materials and methods) 5
一、 細胞培養(Cell culture) 5
二、 質體製備 6
三、 基因轉殖(Transfection) 8
四、 病毒感染(Letiviral infection) 9
五、 蛋白質電泳與西方墨點法(SDS-PAGE and western blotting) 9
六、 免疫螢光染色(Immunofluorescence staining) 10
七、 AKT激酶活性分析(Analysis of AKT kinase activity) 11
參、 結果(Results) 15
一、 分析膀胱尿路上皮細胞癌病患臨床檢體之TMCO1 transcript表現 15
二、 在NOD/SCID異體移植動物模式中過度表現TMCO1抑制腫瘤生長 15
三、 TMCO1經由抑制AKT訊息路徑造成細胞週期停滯 15
四、 TMCO1藉由胺基酸序列S60磷酸化位點增加TP53蛋白質表現量 16
五、 在膀胱尿路上皮細胞癌衍生細胞中TMCO1吸引PHLPP2使AKT1去磷酸化 17
肆、 討論(Discussion) 28
伍、 參考文獻(References) 30
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