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研究生:林佑融
研究生(外文):Yu-Jung Lin
論文名稱:腫瘤缺氧調控叉頭轉錄因子C1促進肺癌之進展
論文名稱(外文):Tumor Hypoxia Regulates Forkhead Box C1 to Promote Lung Cancer Progression
指導教授:陳良築謝佳宏謝佳宏引用關係
口試委員:楊文明陳惠珍吳雨珊吳宗圃
口試日期:2017-03-07
學位類別:博士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:67
中文關鍵詞:腫瘤缺氧缺氧誘導因子-1α肺癌叉頭轉錄因子C1
外文關鍵詞:Tumor hypoxiaHIF-1αLung cancerFOXC1
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目標:叉頭轉錄因子C1具有叉頭結構之DNA結合位,隸屬於叉頭轉錄因子家族。目前有許多的證據指出叉頭轉錄因子C1參與了腫瘤之進展。然而,腫瘤缺氧在叉頭轉錄因子C1對於肺癌的進展中的調控與影響所扮演的角色還並不清楚。
結果:在本篇論文當中,本研究發現叉頭轉錄因子C1在人類或囓齒類的肺癌組織中的缺氧區域是大量表現的。缺氧的環境下可以明顯的誘發叉頭轉錄因子C1的表現。本研究更發現叉頭轉錄因子C1能直接被缺氧誘導因子-1α藉由直接結合在叉頭轉錄因子C1的啟動子上的缺氧結合位所調控。在肺癌細胞中增進叉頭轉錄因子C1的功能能促進其增生,爬行,侵襲,血管新生及上皮-間質轉化的能力。然而,靜默肺癌細胞中叉頭轉錄因子C1的功能反而可以抑制這些現象。更重要的是在小鼠腫瘤模式當中,利用缺氧誘導因子-1α專一性誘導叉頭轉錄因子C1小髮夾RNA而抑制叉頭轉錄因子C1可以抑止腫瘤的生長和血管新生。最後,本研究發現利用脂質奈米粒子包覆經甲基化修飾後的叉頭轉錄因子C1小分子干擾RNA以全身性的給予治療可以抑制腫瘤的生長並延長肺癌模式小鼠之存活率。
結論:總結來說,這些結果指出叉頭轉錄因子C1是一個新穎性的缺氧調控轉錄因子,並且在腫瘤微環境中促進肺癌進展上扮演了一個重要的角色。全身系統性的抑制叉頭轉錄因子C1在肺癌的治療上可能是一個相當有效的治療方式。
關鍵字:腫瘤缺氧、缺氧誘導因子-1α、肺癌、叉頭轉錄因子C1
Purpose: Forkhead box C1 (FOXC1) belongs to the forkhead family of transcription factors which with helix-turn-helix forkhead-like DNA binding domain. More and more reports indicate that FOXC1 is involved in various tumor progressions. However, the mechanism of tumor hypoxia in FOXC1 regulation and its role on lung cancer progression remain unclear.
Results: We find that the expression of FOXC1 was increased in hypoxic areas of lung cancer tissues from rodents or humans. Hypoxia activated FOXC1 transcription through binding of hypoxia-inducible factor-1α (HIF-1α) to the hypoxia-responsive element (HRE) in the FOXC1 promoter via chromatin immunoprecipitation assay. FOXC1 overexpression in lung cancer CL1-0 cells promoted proliferation, migration, invasion, angiogenesis, and epithelial–mesenchymal transition in vitro, whereas FOXC1 shRNA in lung cancer CL1-5 cells inhibited these effects. Moreover, we used specific HIF-1-mediated FOXC1 shRNAs in lung cancer xenograft models to knockdown the tumor hypoxia-induced FOXC1 expression and could suppress tumor growth and angiogenesis through immunofluorescence staining. Finally, systemic delivery of FOXC1 siRNA with 2’-O-methyl modification encapsulated in lipid nanoparticles from tail vein inhibited tumor growth and increased survival time in lung cancer xenograft mice.
Conclusion: These results point out that FOXC1 is a novel hypoxia response gene and plays a critical transcriptional role in tumor microenvironment which promoted lung cancer progression. Systemic delivery FOXC1 siRNA to decrease FOXC1 expression may be an effective therapeutic development for lung cancer.
Keywords: Tumor hypoxia, HIF-1α, Lung cancer, FOXC1
一、 導論 1
二、材料與方法 7
2.1 細胞培養 7
2.2 體外缺氧之方法 7
2.3 質體的構築及慢病毒的傳導 7
2.4 啟動子活性分析方法 10
2.5 染色質免疫沉澱法 11
2.6 西方墨點法 13
2.7 即時定量PCR 15
2.8酵素連結免疫吸附分析法(ELISA) 15
2.9 體外細胞生長試驗 16
2.10 體外細胞爬行及侵襲試驗 16
2.11 體外成管實驗 (Tube formation assay) 17
2.12 動物腫瘤實驗模式 18
2.13 生物性冷光影像系統Bioluminescent imaging (BLI) 19
2.14 FOXC1 siRNA 的修飾及脂質奈米粒子的製作 19
2.15 MicroPET imaging 20
2.16 組織免疫螢光染色 20
2.17 細胞免疫螢光染色 22
2.18 免疫化學組織染色 22
2.19螢光激活細胞分選實驗 23
2.20 統計分析 24
三、結果 25
3.1 FOXC1 在腫瘤缺氧的區域中大量表現 25
3.2 FOXC1 在肺癌病患的檢體中是大量表現並與HIF-1α的表現有關 26
3.3 腫瘤缺氧誘發FOXC1的表現是依賴HIF-1α的存在 28
3.4 HIF-1α 與FOXC1的啟動子結合並調控其表現 29
3.5 FOXC1 促進肺癌細胞的生長以及肺癌的惡化 30
3.6 抑制腫瘤缺氧引起FOXC1的表現能抑止肺癌腫瘤在異種接植小鼠模式上的生長 33
四、討論 36
五、參考文獻 41
六、圖表 49
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