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研究生:呂昱志
研究生(外文):Yu-Zhi Lu
論文名稱:腫瘤微環境下口腔癌細胞粒線體Lon與PYCR1經脯氨酸-膠原蛋白代謝促進細胞外基質之膠原蛋白沉積與癌症轉移之機制探討
論文名稱(外文):Mitochondrial Lon-PYCR1 axis promotes collagen deposition in the extracellular matrix and metastasis in the tumor microenvironment of OSCC via proline-collagen metabolism
指導教授:游偉絢李岳倫李岳倫引用關係
指導教授(外文):Wei-Hsuan YuAlan Yueh-Luen Lee
口試委員:許世明
口試委員(外文):Su-Ming Hsu
口試日期:2023-06-02
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
論文頁數:66
中文關鍵詞:腫瘤微環境癌症轉移膠原蛋白纖維化脯胺酸代謝重組
外文關鍵詞:TMEmitochondriaprolinecollagenmetastasisfibrosis
DOI:10.6342/NTU202300686
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腫瘤微環境調控與影響腫瘤周遭的細胞包括癌相關纖維母細胞 (CAF)、血管內皮細胞、和免疫細胞等等細胞,他們藉由各種生長因子、細胞激素、外基質成分和機械刺激,調控並促進癌症細胞的增生惡化。而粒線體在腫瘤惡化的形成具有重要的影響,當粒線體之代謝失調或是電子傳遞鏈中產生的活性氧自由基 (ROS),促使癌症細胞的慢性發炎和增生。
Lon是粒線體基質中的壓力蛋白,其會受到內質網壓力 (ER stress)、缺氧 (Hypoxia) 和ROS的刺激而活化或表達增加。實驗室先前證實粒線體Lon 會和同位於基質中的PYCR1 形成複合體並調控PYCR1表達量,Lon與PYCR1會促進口腔癌細胞的上皮-間質細胞之轉化 (EMT),PYCR1是一種粒線體酵素,可通過氧化 NADH 合成Proline脯胺酸,Proline約佔膠原蛋白中總氨基酸的 15%以上,細胞外基質 (ECM)中的主要成分之一即為膠原蛋白纖維,當沉積過多會導致腫瘤纖維化。因此,我們首先測定口腔鱗狀上皮癌 (OSCC) 細胞株內之胺基酸代謝,發現粒線體 Lon 和 PYCR1 增加了癌細胞中Proline和Glutamate的含量;於癌症細胞以及纖維母細胞,也觀察到膠原蛋白的表達會受Lon與PYCR1調控而增加。近期的研究也認為,膠原蛋白的增加與腫瘤轉移有很大的相關,而ECM中能夠將膠原蛋白切除的酵素主要為基質金屬蛋白水解酶 (MMP)。因此,本研究也觀察到Lon-PYCR1可以增加 MMP 的表達或活化,例如collagenase (MMP 13), gelatinase (MMP2), and MT-MMP (MMP14),並且活化態會受到ROS影響。我們也發現當 Lon 和 PYCR1表達時,Lysyl oxidase (LOX)的活化態有增加的趨勢。 LOX 被認為是一種缺氧壓力誘發的酵素,促進膠原蛋白硬度與纖維化。歸納上述之證據,本研究證實Lon與PYCR1引起的腫瘤為環境經代謝重組來促使腫瘤纖維化與癌症轉移的趨勢。
Tumor progression depends on a network of surrounding tumor cells, called tumor microenvironment (TME). The TME contains tumor cells and their neighbor cells, including vascular endothelial cells, cancer‑associated fibroblasts (CAFs) and immune cells. The crosstalk within TME involves various growth factors, cytokines, extracellular matrix (ECM) proteins, and mechanical stimulation.
Mitochondria play the important role in tumorigenesis because of the dysregulation of metabolism and Reactive oxygen species (ROS). Lon is a mitochondrial matrix protein and acts as a stress protein induced by ER stress, hypoxia, and ROS. In our previous article, we had validated that Mitochondrial Lon and Pyrroline-5-carboxylate Reductase 1 (PYCR1), a mitochondrial enzyme which synthesizes proline derived from P5C (pyrroline-5-carboxylate), promotes epithelial mesenchymal transition (EMT), cancer metastasis, and inflammation through ROS-involved pathway. Proline, the product of PYCR1, constitutes about 10% of total amino acids in collagen which is the major component in ECM. Hence, we first demonstrated Lon-PYCR1 axis increased metabolism of Proline and Glutamate, and then found the increased synthesis of collagen in OSCC cells and fibroblasts. Moreover, MMP (Matrix Metalloproteinase) is the critical enzyme to degrade collagen fibers and promotes metastasis of cancer cell in ECM. Therefore, we observed that Lon-PYCR1 axis can increase the active forms of MMPs, such as collagenase (MMP-13), gelatinase (MMP-2), and MT-MMP (MMP-14). In addition, we observed the increased active form level of Lysyl oxidase (LOX) when Lon and PYCR1 are over-expressed. LOX is considered as a hypoxia-induced enzyme forming cross-linking in ECM proteins related to collagen stiffness in cancer. Based on the concept of TME, our research dedicated to examine the mechanism of Lon-PYCR1 axis in mitochondrial metabolism and ECM factors promoting fibrosis and metastasis.
口試委員會審定書 i
致謝 ii
中文摘要 iii
英文摘要 iv
Chapter 1. Introduction 1
1.1. Tumor microenvironment (TME) 1
1.2. TME and Reactive oxygen species ROS 1
1.3. Proline in Metabolic reprogramming 2
1.4. Mitochondrial Lon-PYCR1 axis 3
1.5. Collagen synthesis 4
1.6. Lysyl Oxidase and fibrosis 6
1.7. Matrix Metalloproteinase in tumor metastasis 7
Chapter 2. Material and method 10
2.1. Cell culture and cell treatment 10
2.2. Antibodies 11
2.3. Cell lysis 12
2.4. Plasmid 12
2.5. Cell transfection 13
2.6. Western blot 13
2.7. Real Time Quantitative PCR (RT-qPCR) 15
2.8. Soluble collagen assay 16
2.9. Metabolite extraction and Ultra Performance Liquid Chromatography (UPLC) 17
2.10. Immunofluorescence staining 18
2.11. Transwell migration assay and wound healing migration assay 19
Chapter 3. Result 20
3.1. Expression of Mitochondrial Lon and PYCR1 20
3.2. Mitochondrial Lon and PYCR1 regulate Proline and Glutamine / Glutamate Metabolism 21
3.3. Lon and PYCR1 promote the Collagen I and total collagen synthesis but decrease Collagen IV expression in OSCC cell line 22
3.4. Collagen increases in the normal skin fibroblast treated with conditioned medium of cancer cell expressing Lon-PYCR1 24
3.5. Lon and PYCR1 certainly activate the EMT in OSCC cell line and stromal cell 25
3.6. Lon and PYCR1 affect the activation of LOX 26
3.7. Lon and PYCR1 affect the MMP expression and activation 27
3.8. Oxidative stress induced by Lon and PYCR1 increase the expression or active form of MMPs 29
3.9. Lon and PYCR1 promote the migration and invasion ability of OSCC cell line 31
Chapter 4. Discussion 32
4.1. The expression of PYCR1 is regulated by Lon, simultaneously mediating the collagen synthesis 32
4.2. Lon and PYCR1 regulate not only proline metabolism but also Glutamine and Glutamate metabolism 33
4.3. Fibroblast increase the collagen not only because of the Proline provided from cancer cell 34
4.4. LOX acts as a marker of stiffness in fibrotic environment 35
4.5. Lon and PYCR1 induce the ROS-dependent activation of MMPs 35
4.6 Lon and PYCR1 may have different pathway regulating the active form of MMPs 36
4.7 The relationship between collagen deposition and degradation caused by Lon-PYCR1 axis in OSCC cells 37
Chapter 6. Conclusion 39
Chapter 7. Figure 40
Chapter 8. Reference 62
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