臺灣博碩士論文加值系統

先前本實驗室透過肺癌組織定量蛋白體平台發現天門冬胺酸胺醯基化 tRNA 合成酶 (asparaginyl-tRNA synthetase, NARS) 的表現量與非小細胞肺癌之轉移具有高度關聯性。因此想進一步探討 NARS 在非小細胞肺癌癌化過程中所扮演的角色。透過實驗發現在 CL1-0 、 CL1-5 及 H1299肺腺癌細胞株中將 NARS 基因弱化後，細胞的爬行及侵襲能力顯著地下降，然而在另一株 A549 肺腺癌細胞株中卻得到相反的結果。同時我們在細胞生長能力的分析中觀察到將 NARS 基因弱化後，肺腺癌細胞株生長顯著地受到抑制。進一步以流式細胞儀分析發現將 NARS 基因弱化會使 A549 的細胞生長週期會產生延遲以及G1 arrest的現象 。我們也觀察到 NARS 基因弱化後 A549 細胞株中的pAkt 與 pERK 會明顯上升，而在 CL1-5 中的 pAkt 則明顯下降。因此推測在不同的細胞株中 NARS 會透過不同機制調控細胞的爬行與侵襲能力。此外，在 CL1-0 細胞株中利用免疫沈澱法結合質譜儀鑑定到 NARS 交互作用候選蛋白質 HSPA9，並在 CL1-0 、 CL1-5 細胞株中驗證了 NARS 與 HSPA9 的交互作用。最後利用 SILAC 定量蛋白質體策略分析 NARS 基因的弱化後差異性蛋白質的表現，發現 NARS 基因的弱化影響了與細胞骨架以及細胞黏附相關的信息傳遞路徑分子。綜合以上結果，我們推測 NARS 確實參與在肺腺癌細胞轉移以及細胞週期的調控中。

In higher eukaryotic aminoacyl tRNA synthetase (AARs) have been implicated in a variety of noncanonical functions. Previously we identified NARS (asparaginyl-tRNA synthetase) as a potential biomarker for lung adenocarcinoma (ADC) and its overexpression was positively associated with lymph node metastasis in ADC cells. In this study, we found that NARS knockdown reduced the viability, migration and invasion abilities in H1299, CL1-0 and CL1-5 cells but a reverted phenomenon was shown in A549 cells. The growth ability was suppressed in NARS knockdown of ADC cells. Moreover, knockdown of NARS in A549 cells caused the cell cycle arrested in the G1 phase. Knockdown of NARS induced increasing levels of pAkt and pERK in A549 cells, but caused a reduction of pAkt level in CL1-5 cells. We proposed that NARS executes its non-canonical function through protein complex formation, and we identified and verified HSPA9 as an interaction partner of NARS by co-immunoprecipitation combined with GeLC-MS/MS in CL1-0 and CL1-5 cell lines. We also applied stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic strategy combined with NARS knockdown in CL1-5 cells and revealed that NARS protein was involved in cell adhesion cell-matrix interactions and cytoskeleton actin filaments network. Our results collectively suggest that NARS is involved in lung cancer metastasis and regulation of the cell cycle and may though distinct pathway in different ADC cell lines.

指導教授推薦書
口試委員會審定書
誌謝 iii
中文摘要 iv
英文摘要 vi
目錄 vii
表目錄 xiii
圖目錄 xiv
附錄 xvii

第一章 緒論
1.1. 肺癌 (Lung cancer) 1
1.2. 胺醯基化 tRNA 合成酶家族 (Aminoacyl-tRNAsynthetase, ARS) 2
1.3. 天門冬胺酸胺醯基化 tRNA 合成酶 (Asparaginyl-tRNA synthetase, NARS) 7
1.4. 天門冬胺酸胺醯基化 tRNA 合成酶與肺癌的關係 8

第二章 研究目的與策略 9
2.1. 研究目的 9
2.2. 研究目標與策略10

第三章 實驗材料與方法12
3.1. 細胞培養 (Cell culture) 12
3.2. 基因弱化 (Gene knockdown) 13
3.3. 細胞表現質體構築 (Cloning) 14
3.4. 細胞轉染 (Transfection) 19
3.5. 免疫螢光分析 (immunofluorescence assay) 19
3.6. 蛋白質萃取 (Protein extraction) 20
3.7. 蛋白質絕對定量 (Protein Quantitation) 20
3.8. 西方墨點法 (Western blot) 21
3.9. 細胞爬行試驗 (Migration assay) 22
3.10.細胞侵襲試驗 (Invasion assay) 23
3.11.細胞活性試驗 (MTT assay) 24
3.12.細胞週期分析 (Flow Cytometry) 25
3.13.細胞傷痕癒合試驗 (Wound healing) 26
3.14.免疫沈澱 (Immunoprecipitation Assay) 27
3.15.穩定同位素標定與蛋白質鑑定 (SILAC labeling and protein Identification) 28 3.16.銀染 (Silver stain) 29
3.17.膠體內蛋白質水解 (In-gel digestion) 29
3.18.溶液相蛋白質水解 (In-solution digestion) 31
3.19.交互作用網絡分析32

第四章 實驗結果33
4.1. 探討 NARS 對肺腺癌細胞株爬行、侵襲及生長能力的影響. 33
4.2. 探討 NARS 與 Akt 及 ERK 訊息傳導分子的相關性37
4.4. 研究受 NARS 影響之差異性蛋白質與癌化過程的關係42
5.1. 探討 NARS 對肺腺癌細胞株爬行、侵襲及生長能力的 45
5.2. 探討 NARS 與 Akt 及 ERK 訊息傳導分子的相關性47
5.3. 找尋 NARS 交互作用蛋白質及探討 NARS 與其交互作用蛋白質之相關49
5.4. 研究受 NARS 影響之差異性蛋白質與癌化過程的關係52

參考文獻57
附錄93

圖表65

表目錄
表ㄧ. NARS 之雙股小片段 RNA 序列65
表二. NARS 引子設計66
表三. NARS 基因弱化後具 1.5 倍差異性表現之分子可能參與之 pathway map 及 process Networks 67
表四. 肺腺癌細胞株基因組態68

圖目錄
圖ㄧ. 在肺腺癌細胞株中探討 NARS-mediated 肺癌轉移的實驗策 略69
圖二. 在四株肺腺癌細胞株中將 NARS 基因弱化後觀察其細胞爬行 能力試驗 (Migration Assay) 70
圖三. 在兩株肺腺癌細胞株將 NARS 基因弱化後其細胞群聚爬行能 力 ( Wound-healing Assay ) 71
圖四. 在四株肺腺癌細胞株將 NARS 基因弱化後其細胞侵襲能力試 驗 (Invasion Assay) 72
圖五. 在三株肺腺癌細胞株將 NARS 基因弱化後細胞生長能力分 析 73
圖六. 在三株肺腺癌細胞株 NARS 基因弱化後細胞週期分 析74
圖七. 在 Hela 及 A549 細胞株過度表現 NARS /pcDNA3.1A 載 體78 圖八. 在三株肺腺癌細胞將 NARS 基因弱化對磷酸化 Akt 含量的影響79
圖九. 在三株肺腺癌細胞將 NARS 基因弱化對磷酸化 ERK 含量的 影響80
圖十. 在 CL1-0 肺腺癌細胞株以 NARS 抗體進行免疫沈澱尋找與 NARS 交互作用之蛋白質81
圖十一. NARS 交互作用之候選蛋白質82
圖十二. 以西方墨點法結合免疫沈澱法驗證 HSPA9 為 NARS 之交 互作用蛋白質83
圖十三. 在肺腺癌病人周邊正常組織與腫瘤組織檢體中 NARS 與 HSPA9 的表現量與相關性84
圖十四. 在 oncomine 肺腺癌數據庫中正常組織與腫瘤組織檢體中 NARS 與 HSPA9 的表現量與相關性85
圖十五. 利用 SILAC labeling 結合蛋白質體學鑑定分析肺腺癌細胞株在 NARS 基因弱化後的差異性蛋白質表現86
圖十六. NARS 基因弱化後的差異性蛋白質主要受 c-Myc 及 CREB1 轉錄因子所調控87
圖十七. 利用西方墨點法驗證 NARS 可能參與之訊息傳遞路徑中的候選蛋白質91
圖十八. 整合 NARS 具交互作用的蛋白質資料庫及 NARS 基因弱化後具差異性表現的蛋白質資料庫中篩選出與肺癌癌化相關分子92

附錄目錄

附圖一. 胺醯基化 tRNA 合成酶典型功能93
附圖二. 催化轉譯作用無關之新結構域94
附圖三. 肺腫瘤組織定量蛋白體平台偵測 NARS 的表現量95
附圖四. Human Protein Atlas 資料庫中 NARS 在腫瘤組織中的表 現96
附圖五. NARS Construct Map 97
附圖六. Human Protein Atlas 資料庫中 FLG2 在人類個組織中的表現量98
附圖七. Classical phosphoinositide 3 kinase (PI3K)/Akt signaling pathway 99
附圖八. The activation of PI3K/Akt/NF-κB pathway on NSCLC invasion 100
附圖九. Schematic overview of MAPK pathways 101

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