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研究生：	黃永蒼 Huang, Yung-Tsang
論文名稱：	嗜中性白血球明膠酶相關脂質運載蛋白NGAL在肺癌進展過程調控第九型基質金屬蛋白酵素MMP-9的活性 Neutrophil gelatinase-associated lipocalin modulates Matrix metalloproteinase-9 activity in lung cancer progression
指導教授：	洪建中 Hung, Jan-Jong
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物資訊與訊息傳遞研究所 Insitute of Bioinformatics and Biosignal Transduction
論文出版年：	2017
畢業學年度：	105
語文別：	中文
論文頁數：	60
中文關鍵詞：	MMP-9 、NGAL 、雙硫鍵、轉移、肺癌
外文關鍵詞：	NGAL, MMP-9, Disulfide bond, Metastasis, Lung cancer
相關次數：	點閱：344 下載：3
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在台灣肺癌的患者一直都是癌症十大死因之冠，盡管現在有化學藥物療法與標靶藥物，也有新開發的免疫療法來治療並減緩病情，但肺癌的五年存活率仍不到百分之十五。至目前為止主要肺癌造成死亡的主因，是肺癌轉移，癌轉移造成其他器官或組織形成轉移的肺癌細胞；主要癌轉移會透過癌細胞分泌出基質金屬蛋白酵素 (Matrix metalloproteinases, MMPs) 去分解細胞外基質，而造成癌細胞進入到循環系統，再轉移到其他器官或組織形成癌轉移；在分解細胞外基質時，癌細胞主要會分泌許多種基質金屬蛋白酵素 (MMPs)，其中第九型基質金屬蛋白酵素 (MMP-9) 最被重視及廣泛研究，這些酵素會造成癌細胞的侵犯能力增強。但會影響MMP-9活性且與MMP-9交互作用的蛋白質如嗜中性白血球明膠酶相關脂質運載蛋白 (Neutrophil gelatinase-associated lipocalin, NGAL) 的機制其實還有許有未解開的謎團。NGAL是一個運載蛋白，最原本的在病原菌感染時，NGAL會藉由抓住鐵離子的siderophere來抑制病原菌的生長，但過去也有許多研究發現在癌症中，NGAL往往是過度表現的狀態，影響了發炎、癌細胞的轉移等等。我們首先發現NGAL在低濃度的狀況下，會影響肺癌細胞的移動與侵犯能力，但不影響增生能力。而除了過去研究推測MMP-9會藉由雙硫鍵與NGAL交互作用外，因為老鼠的Lcn2在序列上與NGAL上的Cysteine 107的序列並不同，我們也藉由不同物種，發現老鼠的Lcn2 (同NGAL) 與MMP-9之間也有交互作用，藉此告訴我們NGAL中的Cysteine與MMP-9交互作用且形成複合物的能力各有不同。
所以我們接著以不同突變型分別為C96V、C107V、C195V、雙突變與參突變的NGAL，發現在Cysteine 195對於MMP-9交互作用所形成複合物的比例增加許多，且參突變的NGAL是不會與MMP-9交互作用的。在過去也指出當MMP-9與NGAL形成複合物後，會讓MMP-9影響癌細胞的移動能力與侵犯能力也會增強，所以我們進一步發現以突變的NGAL與MMP-9形成的複合物直接處理肺癌細胞，NGAL Cysteine 195突變型除了會抑制NGAL原本影響的侵犯能力，也降低MMP-9所給予增加肺癌細胞的移動與侵犯能力。
對於突變型的NGAL在活體內的實驗目前還在進行當中，但對於抑制肺癌轉移的影響，我們的確在肺癌細胞內實驗看到了明顯降低的作用，也許對於未來開發藥物或設計新的蛋白質抑制物，突變型的NGAL都可能是一個重要且具潛力的標的蛋白，且提高肺癌患者存活率的新治療策略。

Matrix metalloproteinase-9 (MMP-9) is one of crucial proteinases involved in cancer metastasis through the degradation of extracellular matrix. Although the neutrophil gelatinase-associated lipocalin (NGAL) /MMP-9 complex is increased in malignant cancers, the role of NGAL in MMP-9-mediated cancer malignancy still needs to be elucidated. NGAL involve in the innate immune system to limit pathogen proliferation, while NGAL also highly expresses in malignant cancers. In this study, the role of NGAL in cancer formation was studied in vitro and in vivo. In in vitro experiments indicated that NGAL can’t alter the cell proliferation. On the other hand, in in vivo data show that NGAL treatment inhibits tumor formation, implying that NGAL might modulate tumor-associated microenvironment to repress lung cancer formation in vivo. Next, we studied the interaction mechanism(s) between NGAL and MMP-9. Since both of NGAL and MMP-9 contain many disulfide bonds. We hypothesized that inter- or intra-molecular disulfide bonds might play an important role in regulating the interaction of NGAL and MMP-9. Therefore, various mutants were built in NGAL including C96V, C107V, C195V, double mutation and triple mutation for studying the interaction between NGAL and MMP-9. Data indicated that NGAL-C96V and NGAL-C96V/C107V increase the NGAL dimerization and NGAL/MMP-9 complex, suggesting that loss of C96 or C107 thereby unveil C195 is important for NGAL dimerization and NGAL/MMP-9 complex formation. Finally, the effects of the NGAL and its mutants in MMP-9-mediated metastasis were characterized. All the results in migration and invasion assays reveal that loss of C195, NGAL-C195V, dramatically repress the metastasis ability. In conclusion, NGAL secreted out of neutrophil is involved in lung cancer progression through the interaction with MMP-9, and residue, C195, of NGAL might play a crucial role. NGAL-C195V can be as a potential candidate for development of cancer drug in the future.

目錄
中文摘要 ................................................I
英文摘要	..............................................III
誌謝.................................................VIII
目錄....................................................X
圖目錄.................................................XI
緒論....................................................1
第一節	前言............................................1
第二節	肺癌 (Lung cancer)..............................1
第三節 遠端轉移..........................................2
第四節 基質金屬蛋白酶 (Matrix metalloproteinases, MMPs)...3
第五節 第九型基質金屬蛋白酶 (Matrix metalloproteinase-9)..3
第六節 嗜中性白血球明膠酶相關脂質運載蛋白(Neutrophil gelatinase-associated lipocalin)........................5
第七節 NGAL/MMP-9複合物的機制............................6
第八節 研究動機..........................................7
實驗材料.................................................8
實驗方法................................................12
實驗結果................................................24
第一節 NGAL不影響肺癌細胞的增生能力但減少肺部腫瘤的形成....24
第二節 NGAL與MMP-9在肺癌細胞侵襲、移動能力與表現的相關性...24
第三節 NGAL與MMP-9形成複合物的詳細機制...................25
第四節 人類NGAL與老鼠Lcn2之間的差異性與MMP-9構成複合物....25
第五節 NGAL中的Cysteine參與NGAL二聚體的形成與NGAL/MMP-9 複合物的形成................................................26
第六節 野生型與突變型NGAL與NGAL/MMP-9 複合物影響MMP-9活性.27
第七節 Conditioned medium影響肺癌細胞移動的能力..........28
討論...................................................30
第一節 MMP-9與NGAL形成複合物與二聚體的關係................30
第二節 老鼠的Lcn2 影響肺癌細胞的移動能力..................30
第三節 NGAL在肺癌中的角色................................31
第四節 Conditioned medium 不影響EMT指標蛋白..............31
第五節 新穎性...........................................32
參考文獻................................................33
圖目錄
Figure 1. NGAL had no effect in proliferation, but affected morphology in lung cancer cells. (human NGAL).37
Figure 2. NGAL protein treatment promoted the migration ability in lung cancer cells...........................38
Figure 3. NGAL not only affect the migration ability, but also increase the invasive ability in lung cancer cell.40
Figure 4. NGAL can upregulation MMP-9 protein expression level slightly in lung cancer cell.....................41
Figure 5. HA-tag of MMP-9 and MMP-9 can interact with NGAL in lung cancer cell...............................42
Figure 6. MMP-9L (truncated form of N terminal) can interact with NGAL in vitro............................43
Figure 7. Lcn2 (mouse) also can interact with MMP-9 in lung cancer cell.......................................44
Figure 8. The Cysteine sites of NGAL protein...........45
Figure 9. The dimer formation of mutated NGAL protein..46
Figure 10. MMP-9, NGAL and mutated NGAL composed complexes in conditioned medium........................47
Figure 11. The complexes formation and MMP-9 activities of MMP-9, MMP-9/ NGAL and MMP-9/mutated NGAL in conditioned medium.....................................49
Figure 12. The conditioned medium of MMP-9, MMP-9/ NGAL and MMP-9/mutated NGAL affected lung cancer cell migration abilities....................................51
Figure 13. The conditioned medium of MMP-9, MMP-9/ NGAL and MMP-9/C195V NGAL affected lung cancer cell migration abilities..............................................52
Figure 14. The conditioned medium of MMP-9, MMP-9/ NGAL and MMP-9/C195V NGAL affected lung cancer cell invasive abilities..............................................53
Figure 15. NGAL decreased the lung tumor volume in B6 SCGB1A1-rtTA drove EGFRL858R mice......................55
Appendix 1. Pro-MMP-9 (truncated form of N-terminal) can interact with NGAL, the Pro-MMP-9/NGAL complex not only constitute monomer to monomer, but also compose of dimer to monomer.............................................56
Appendix 2. Lcn2 increase migration ability in human lung cancer cell. (mouse NGAL)..............................57
Appendix 3. The complexes formation and MMP-9 activities of MMP-9, MMP-9/ NGAL and MMP-9/mutated NGAL in conditioned medium.....................................58
Appendix 4. The protein expression of EMT marker did not increase a lot in the conditioned medium of MMP-9, MMP-9/ NGAL and MMP-9/mutated NGAL in lung cancer cell........59
Appendix 5. The system of SCGB1A1-rtTA drove EGFRL858R mice induced lung tumor by fed doxycycline six weeks...60



                                    

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