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研究生:張御展
研究生(外文):CHANG, YU-CHAN
論文名稱:葡萄糖運輸蛋白4透過TRIM24訊息傳遞路徑調控頭頸部腫瘤轉移
論文名稱(外文):Glucose transporter 4 promotes head and neck squamous cell carcinoma metastasis through TRIM24-DDX58 axus
指導教授:蕭宏昇
指導教授(外文):Hsiao, Michael
口試委員:謝義興蕭宏昇劉亞平黃世明林源峰吳駿翃張牧新
口試委員(外文):Shieh, Yi-ShinHsiao, MichaelLiu, Ya-PingHuang, Shih-MingLin, Yuan-FengWu, AlexChang, Peter Mu-Hsin
口試日期:2017-03-06
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
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:117
中文關鍵詞:頭頸部腫瘤轉移葡萄糖運輸蛋白4
外文關鍵詞:Head and neck squamous cell carcinomametastasisglucose transporter 4
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頭頸部鱗狀上皮細胞癌(Head and Neck Squamous Cell Carcinoma, HNSCC)是一種獨特且主要的全球性癌症之一。先前研究指出頭頸部鱗狀上皮細胞對於葡萄糖攝取和糖解作用皆會顯著增加。其中,葡萄糖運輸蛋白(Glucose Trasnporter, GLUT)扮演著糖解途徑(glycolysis)中的主要樞紐,其中GLUT4具有最高的葡萄糖親和力。然而,GLUT4在頭頸部鱗狀上皮細胞癌中的詳細作用機制還沒完全研究透徹。首先我們在頭頸部鱗狀上皮細胞癌的臨床族群中,利用進行電腦模擬分析,鑑定出GLUT4為最重要的葡萄糖運輸蛋白且與臨床患者有顯著的預後相關性。我們也蒐集了90個頭頸部鱗狀上皮細胞癌臨床病患檢體來製作組織微陣列的免疫組織化學分析。從臨床病理分析發現了GLUT4的過度表達與頭頸部鱗狀上皮細胞癌的整體存活率(overall survival, OS, P=0.035)與腫瘤復發(recurrence-free survival, RFS, P=0.001)有高度相關性。此外,在體外或體內模型皆觀察到若過度表現GLUT4在低內原性的腫瘤細胞中,會導致遷移能力顯著增加。反之,我們亦觀察到調降GLUT4表現在高內原性的腫瘤細胞中會得到反向功能功能表型。進一步的,我們建立了GLUT4過度表達模型之DNA微陣列晶片,並透過途徑分析軟體(Ingenuity pathway analysis, IPA)分析其潛力參與調控的轉錄因子,我們確立了TRIM24為GLUT4主要下游的調節因子並透過更下游的標靶DDX58來活化GLUT4-TRIM24路徑引起的遷移能力。透過這些發現,我們確定葡萄糖代謝變化在頭頸部鱗狀上皮細胞癌進展且轉移能力增強有部分依賴新發現的GLUT4-TRIM24訊息傳遞路徑。此新路徑未來也許能應用於頭頸部鱗狀上皮細胞癌預後和標靶治療上。
Metabolic reprograming and Warburg effect as an important issue in head and neck squamous cell carcinoma (HNSCC) involved drug-resistance, tumorigenesis and tumor metastasis. Solute Carrie family 2, also called glucose transporter family members play an initial role in glycolysis pathway by controlling transporter efficiency. However, detail mechanism of GLUT family in HNSCC tumorigenesis has not been fully investigated. In our study, GLUT4 had most significant value compared with other family members associating with HNSCC patients’ survival rate. Furthermore, we found the GLUT4 protein level positive correlated with N status by immunohistochemistry analysis. The results showed that increased GLUT4 expression level was associated with poor overall survival (OS, P=0.035) and recurrence-free survival (RFS, P=0.001). We further confirmed that GLUT4 could promote HNSCC migration and invasion in vitro and in vivo through two-way complementary model including GLUT4 overexpression or knockdown stable cell models. We established microarray chips to investigate the detail molecular mechanism. Ingenuity Pathway Analysis (IPA) software dissected tripartite motif-containing 24 (TRIM24) transcription factor as the top ranking downstream regulator of GLUT4. DDX58 was further validated that the major downstream target of GLUT4-TRIM24 axis in HNSCC. Knockdown of DDX58 could reverse the phenotype included migration and invasion by GLUT4-TRIM24 activation in our model. Combined all evidence, GLUT4/TRIM24/DDX58 could be an independent prognostic marker for HNSCC patients. These findings may a novel therapeutic development to combat metastasis of HNSCC in the future.
正文目錄
緒言 1
材料與方法 13
1.細胞培養 13
2.基因持續表現載體及病毒製備 13
3.西方墨點法 (Western blot) 13
5.葡萄糖攝取和乳酸生成 15
6.細胞遷徙/侵襲 15
7.反轉錄聚合酶連鎖反應 16
8.試劑 16
9.免疫組織染色(Immunohistochemistry staining) 16
10.動物模型 17
11.病例 18
12.統計學分析 18
結果 20
GLUT4表現量增加與頭頸部腫瘤轉移及病患預後差有高度顯著相關性 20
GLUT4不正常的過度表現促進頭頸部腫瘤細胞的遷徙和侵襲能力 22
GLUT4表現增加促進活體模型的肺轉移和局部頸部淋巴侵襲 23
GLUT4過度表現所誘導的頭頸部腫瘤轉移具有葡萄糖解作用非依賴性 24
GLUT4透過TRIM24-DDX58訊息路徑調控頭頸部腫瘤轉移 25
討論 27
結論 31
參考文獻 32
表 1. GLUT4蛋白表現與臨床參數相關分析表 a
表 2. 單因素和多因素分析頭頸部腫瘤臨床患者之GLUT4表現量 b
表3. 頭頸部腫瘤臨床病患之相關人口統計學分析 c
表4. GLUT4過度表現所活化的潛在轉錄因子及其下游目標(根據Z-score排序) d
表5. GLUT4過度表現所抑制的潛在轉錄因子及其下游目標(根據Z-score排序) e
表6. TRIM24 下游基因及其表現量列表 f
表7. 引子序列及載體資訊 g
表8. 表現量兩倍以上於GLUT4過度表現細胞與控制組相比之探針列表 h
附錄 a
縮寫表 a
過度表現載體圖譜 b
調降表現載體圖譜 c
表目錄
表 1. GLUT4蛋白表現與臨床參數相關分析表 a
表 2. 單因素和多因素分析頭頸部腫瘤臨床患者之GLUT4表現量 b
表3. 頭頸部腫瘤臨床病患之相關人口統計學分析 c
表4. GLUT4過度表現所活化的潛在轉錄因子及其下游目標(根據Z-score排序) d
表5. GLUT4過度表現所抑制的潛在轉錄因子及其下游目標(根據Z-score排序) e
表6. TRIM24 下游基因及其表現量列表 f
表7. 引子序列及載體資訊 g
表8. 表現量兩倍以上於GLUT4過度表現細胞與控制組相比之探針列表 h


圖目錄
圖1. Heat-map 顯示葡萄糖運輸蛋白家族的mRNA表現量與頭頸部腫瘤是否發生轉移之間的相關性 a
圖2. GLUT4在高度危險群組中表現量較高且與預後差有高度顯著相關性 b
圖3. GLUT1, GLUT2和GLUT3根據RNA表現量定義高低後分析臨床頭頸部腫瘤患者生存率在Petel臨床資料庫 c
圖4. GLUT5, GLUT6和GLUT8根據RNA表現量定義高低後分析臨床頭頸部腫瘤患者生存率在Petel臨床資料庫 d
圖5. GLUT9, GLUT10和GLUT11根據RNA表現量定義高低後分析臨床頭頸部腫瘤患者生存率在Petel臨床資料庫 e
圖6. GLUT12, GLUT13和GLUT14根據RNA表現量定義高低後分析臨床頭頸部腫瘤患者生存率在Petel臨床資料庫 f
圖7. 流行病學統計所有葡萄糖運輸蛋白RNA表現與相對應的危險因子(Hazard ratio)於頭頸部腫瘤族群中 g
圖8. GLUT4免疫組織染色 h
圖9. GLUT4組織免疫化學染色之評分標準 i
圖10. GLUT4組織免疫化學分析與臨床病患存活率(整體存活率/腫瘤復發存活率) j
圖11. 分析8株頭頸部腫瘤細胞中GLUT4蛋白質表現量 k
圖12. 分析8株頭頸部腫瘤細胞中細胞遷徙/侵襲能力 l
圖13. 分析8株頭頸部腫瘤細胞中細胞遷徙/侵襲能力之量化圖 m
圖14. 分析8株頭頸部腫瘤細胞中GLUT4 蛋白表現量與細胞侵襲能力之相關性 n
圖15. 在HSC-3和FaDu細胞中建立GLUT4過度表現模型 o
圖16. GLUT4過度表現模型之功能性分析與量化圖 p
圖17. 在HSC-3-M3和HSC-2細胞中建立GLUT4調降模型 q
圖18. GLUT4調降模型之功能性分析與量化圖 r
圖19. 透過GLUT4過度表現FaDu細胞株建立小鼠肺轉移模型與量化 s
圖20. 透過GLUT4過度表現FaDu細胞株建立小鼠原位腫瘤模型 t
圖21. 透過GLUT4過度表現FaDu細胞株建立小鼠原位腫瘤模型之平均訊號量化圖 u
圖22. 分析FaDu細胞中過度表現GLUT4之生長速率 v
圖23. GLUT4過度表現細胞在FaDu細胞中透過小鼠皮下腫瘤 (上圖)以及腫瘤重量(下圖)評估生長速率 w
圖24. 相對量化8株頭頸部腫瘤細胞之葡萄糖吸收率以及乳酸產生速率 x
圖25. GLUT4過度表現模型與2-NBDG吸收率以及抑制劑Ritonavir加入組別相關量化圖 y
圖26. 抑制劑ritonavir和indinavir加入於GLUT4過度表現細胞分析葡萄糖攝取率之量化圖 z
圖27. 抑制劑ritonavir和indinavir加入於GLUT4過度表現FaDu細胞分析細胞遷徙/侵襲能力與量化圖 aa
圖28. 抑制劑ritonavir和indinavir加入於GLUT4過度表現HSC-3細胞分析細胞遷徙/侵襲能力與量化圖 bb
圖29. IPA軟體分析GLUT4過度表現與控制組相比細胞之潛在活化/抑制的轉錄因子 cc
圖30. GLUT4 過度表現結合IPA軟體內建TRIM24與其下游目標平臺 dd
圖31. 分析GLUT4 過度表現模型中TRIM24下游因子DDX58與OASL蛋白質變化 ee
圖32. 分析GLUT4 調降表現模型中TRIM24下游因子DDX58與OASL蛋白質變化 ff
圖33. 分析GLUT4 調降表現模型中加入DDX58以及OASL 相對應之shRNAs後TRIM24下游因子DDX58, OASL蛋白質變化 gg
圖34. DDX58以及OASL 相對應之shRNAs加入GLUT4 調降表現模型進行細胞遷徙能力分析 hh
圖35. DDX58以及OASL 相對應之shRNAs加入GLUT4 調降表現模型進行細胞遷徙能力分析量化圖 ii
圖36. DDX58或OASL 與GLUT4 表現高低合併成預後指標來計算臨床頭頸部腫瘤患者之整體存活率 jj
圖37. 於頭頸部腫瘤臨床患者檢體中運用GLUT4和OASL的RNA表現量進行相關性分析 kk
圖38. 於頭頸部腫瘤臨床患者檢體中運用GLUT4和DDX58的RNA表現量進行相關性分析 ll
圖38. 於頭頸部腫瘤臨床患者檢體中檢測GLUT4 基因變異比例 mm


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