基質金屬蛋白酶 1 在酸性胰臟腫瘤微環境中的功能性探討

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研究生：	陳羽萱 Chen, Yu-Hsuan
論文名稱：	基質金屬蛋白酶 1 在酸性胰臟腫瘤微環境中的功能性探討 Roles of Matrix Metalloproteinase 1 (MMP-1) in Acidic Pancreatic Tumor Microenvironment
指導教授：	張壯榮 Chang, Chuang-Rung 張文祥 Chang, Wun-Shiang
口試委員:	王翊青 Wang, I-Ching 林愷悌 Lin, Kai-Ti
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	79
中文關鍵詞：	基質金屬蛋白酶、MMP-1 、酸性腫瘤微環境、細胞外酸鹼值、pHe 、胰臟癌
外文關鍵詞：	matrix metalloproteinase, MMP-1, acidic tumor microenvironment, extracellular pH, pHe, pancreatic cancer
相關次數：	點閱：1 下載：0
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酸化微環境為許多實體腫瘤之共同特徵。在胰臟癌病患體內，其實體腫瘤內部血管灌注不足，導致腫瘤細胞所需之氧氣和養分供應缺乏。這些腫瘤細胞喜好優先使用糖解作用而不是氧化磷酸化的方式去產生能量，此現象稱為瓦式效應。然而，伴隨此瓦式作用，癌細胞會產生大量酸性代謝物質並迅速將之分泌至細胞外，終使胰臟腫瘤胞外酸鹼值 (extracellular pH, pHe) 逐漸失衡且變質變酸。藉由本論文的實驗研究，我發現當胰臟腫瘤處於外在環境變酸的壓力刺激下，癌細胞會大量上調「基質金屬蛋白酶-1 (MMP-1)」的表現量，且其增加之基因轉錄倍數，在人體所有二萬多筆基因數當中排名最高。進一步分析顯示，酸化環境誘導MMP-1的大量表現並被分泌至胞外，進而促使胰臟腫瘤細胞變得更加惡化且更具侵襲與轉移能力。這些研究發現除了點出MMP-1蛋白酶於酸性腫瘤微環境中的可能重要功能，亦提供了未來針對胰臟腫瘤的外在偏酸特性設計基質蛋白酶-1抑制劑的抗癌策略方針。

Acidic microenvironment is a hallmark of many solid tumors. In pancreatic cancer patients, their solid tumors are often provided with poor vascular perfusion which leads to the disrupted flow of oxygen and nutrients to the tumor mass. These tumor cells preferentially utilize glycolysis instead of oxidative phosphorylation, a phenomenon known as Warburg effect. However, due to exuberant secretion of acidic metabolites from Warburg metabolism, the extracellular pH (pHe) of pancreatic tumors generally becomes acidic per se. In this thesis, I present evidence showing that matrix metalloproteinase 1 (MMP-1) has the highest transcriptional fold increase of the whole transcriptome (more than 25,000 genes for human genome) upon tumor exposure to acidosis stress. The increased expression and subsequent secretion of MMP-1 induced by extracellular acidity was further identified to promote pancreatic tumor malignancy and aggressiveness. These findings not only highlight the important roles of MMP-1 under the acidic tumor microenvironment, but also suggest that future development of acidity-targeting MMP-1 inhibitor may provide a rational therapeutic approach to the treatment of pancreatic cancer.

CONTENTS
Abstract (in English) I
Abstract (in Chinese) II
Acknowledgement III

CHAPTER 1     GENERAL INTRODUCTION
1.1    Pancreatic Cancer 2
1.2    Acidic Microenvironment of Pancreatic Cancer 2
1.3    Matrix Metalloproteinases in Pancreatic Cancer 3
1.4    Potential Role of Matrix Metalloproteinase 1 (MMP-1)
in Acidic Pancreatic Tumor Microenvironment 5

CHAPTER 2     MATERIALS AND METHODS
2.1     Materials
2.1.1     Cell Culture 7
2.1.2     MTT Assay 7
2.1.3     RNA Extraction and Real-Time PCR 8
2.1.4     Western Blotting 9
2.1.5     Gene Knockdown 13
2.1.6     ELISA 13
2.1.7     Zymography 13
2.1.8     Transwell Migration Assay 14
2.1.9     Transwell Invasion Assay 15
2.1.10     ChIP Assay 15
2.1.11     Mitochondria Labelling 16
2.2     Methods
2.2.1     Cell Culture 16
2.2.2     MTT Assay 17
2.2.3    Real-Time RT-PCR 17
2.2.4     Western Blotting 18
2.2.5     Gene Knockdown 19
2.2.6     ELISA 20
2.2.7     Zymography 20
2.2.8     Transwell Migration Assay 20
2.2.9     Trranswell Invasion Assay 21
2.2.10     ChIP Assay 21
2.2.11    Mitochondria Labelling 22
CHAPTER 3     RESUITS
3.1  Effects of Pancreatic Cancer Cells Under Acidic pHe 24
3.2  MMP-1 Expression under Acidic Extracellular Stress 26
3.3  Potential transcriptional regulation of MMP-1 in response
to acidic pHe 28
3.4  Effects of MMP-1 in Pancreatic Tumor Cells under
Acidic Environmental Stress 29

CHAPTER 4    DISCUSSION
4.1     Discussion 33

REFERENCES 35

TABLES 43

FIGURES 48

APPENDIXES 76
 
INDEX OF TABLES

Table 1.      Transcriptional fold changes of the matrix metalloproteinase family upon acidic pHe treatment 44

Table 2.      Transcriptional fold changes of the cathepsin proteinase family upon acidic pHe treatment 46

Table 3.      Transcriptional fold changes of the transcription factors upon acidic pHe treatment 47

 
INDEX OF FIGURES

Figure 1.     The acidified extracellular microenvironment — a hallmark of solid tumors 49
Figure 2.     Schematic diagram of overall experimental design in this thesis 50
Figure 3.     Morphology of SUIT-2 pancreatic cancer cells cultured in  the control (pHe 7.4), buffer (pHe 7.4) or acidic (pHe 6.7) medium 51
Figure 4.     The proliferation rate of SUIT-2 pancreatic cancer cells cultured in either the control (pHe 7.4), buffer (pHe 7.4) or acidic (pHe 6.7) medium 52
Figure 5.     Cell tracking assay of SUIT-2 pancreatic cancer cells cultured in either the control (pHe 7.4), buffer (pHe 7.4) or acidic (pHe 6.7) medium 53
Figure 6.     The DQ-collagen IV assay of SUIT-2 pancreatic cancer cells cultured in either the control (pHe 7.4), buffer (pHe 7.4) or acidic (pHe 6.7) medium 54
Figure 7.         Schematic diagram of results of SUIT-2 pancreatic cancer cells under acidic pHe 55
Figure 8.     Quantitative RT-PCR of endogenous MMP-1 expression of SUIT-2 pancreatic cancer cells cultured in either the control (pHe 7.4), buffer (pHe 7.4) or acidic (pHe 6.7) medium 56
Figure 9.     Extracellular acidic pHe activates the expression of MMP-1 in SUIT-2 pancreatic cancer cells 57
Figure 10. ELISA assay to evaluate the secretion of MMP-1 in SUIT-2 pancreatic cancer cells cultured in either the control (pHe 7.4), buffer (pHe 7.4) or acidic (pHe 6.7) medium 58
Figure 11. Time-dependent expression of MMP-1 in SUIT-2 pancreatic cancer cells under acidic pHe 59
Figure 12. Expression of NF-κB2 in SUIT-2 pancreatic cancer cells cultured in either the control (pHe 7.4), buffer (pHe 7.4) or acidic (pHe 6.7) medium 61
Figure 13. Expression of MMP-1 of SUIT-2 pancreatic cancer cells cultured in Ctrl, buffer, and acidic conditioned medium for a week and transfected with NC siRNA and NF-κB2 siRNA under acidic pHe 62
Figure 14. ChIP-assay of NF-κB2 binding to the MMP-1 promoter region of the SUIT-2 pancreatic cancer cell line 64
Figure 15. Zymography analysis of MMP-1 enzymatic activity 66
Figure 16. Knockdown of MMP-1 in SUIT-2 pancreatic cancer cells cultured in the acidic (pHe) medium 67
Figure 17. Morphological analysis of SUIT-2 pancreatic cancer cells transfected with NC siRNA or MMP-1 siRNA under acidic pHe 69
Figure 18. The proliferation rate of SUIT-2 pancreatic cancer cells transfected with NC siRNA or MMP-1 siRNA under acidic pHe 70
Figure 19. Migration assay of SUIT-2 pancreatic cancer cells transfected with NC siRNA or MMP-1 siRNA under acidic pHe 71
Figure 20. Invasion assay of SUIT-2 pancreatic cancer cells transfected with NC siRNA or MMP-1 siRNA under acidic pHe 72
Figure 21. Mitochondrial distribution of SUIT-2 pancreatic cancer cells transfected with NC siRNA or MMP-1 siRNA under acidic pHe 73
Figure 22. Summary of overall findings 75

INDEX OF APPENDIXES

Appendix 1. Expression levels of MMP-1 protein in BxPC3, MDA-MB-231 and NCI-
H1299 cell lines upon acidic pHe 77
Appendix 2. Expression levels of MMP-1 protein in MIA PaCa-2 and PANC-1
pancreatic cancer cell lines under acidic pHe 78
Appendix 3. Autophagy LC3B protein expression levels was observed after MMP-1
knock-down in SUIT-2 pancreatic cancer cells under acidic pHe 79


                                

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