腫瘤的發展與癌細胞因應腫瘤微環境的壓力進行代謝重新編有關。Lon 是一種位於粒線體基質的蛋白酶，可幫助癌細胞對不利微環境，如高氧化壓力、缺氧等，做出反應以利存活。粒線體在細胞內擔任能量工廠、產生ATP，呼吸鏈與許多代謝生化反應在此胞器進行。先前研究發現，Lon 可以通過促進上皮間質轉型的方式來誘導癌細胞發生轉移作用，並且可與pyrroline-5-carboxylate reductase 1 (PYCR1) 結合，影響其穩定性。PYCR1同樣也是一種位在粒線體的酵素，負責脯氨酸的合成。之前的研究認為，脯氨酸的生成會促進膠原蛋白合成，並使細胞外基質硬化而促進癌症轉移。先前也已經證實Lon與PYCR1有交互作用，然而，Lon 如何藉由與 PYCR1的相互作用來調節脯氨酸相關的代謝重新編程和轉移，其詳細機制尚未了解。在本研究中，發現當癌症細胞 Lon 過量表達時，PYCR1 的蛋白含量會升高，並且Lon透過上調PYCR1的作用促進膠原蛋白合成，而可能使細胞外基質硬化，從而誘導腫瘤轉移。此外，Lon與PYCR1增加了NAD+/NADH的比率，這可能是相互作用下改變了細胞代謝的形式，而進一步促進瓦式效應的機制。綜上所述，本研究表明經Lon與PYCR1的相互作用影響代謝重新編程，進而支持癌細胞增殖和轉移作用。

Tumor progression is associated with metabolic reprogramming in the tumor microenvironment (TME). Lon is a mitochondrial protease, helps cancer cells response to the stress, such as peroxidation stress, hypoxic. Besides, Lon induces cancer cell metastasis by promotes epithelial-mesenchymal transition (EMT). Similarly, pyrroline-5-carboxylate reductase 1 (PYCR1) is a mitochondrial enzyme which is essential for synthesis of proline. Many studies demonstrated that proline metabolism is also related to cancer metastasis by increase collagen synthesis to making extracellular matrix (ECM) stiffening. Previous study has showed that Lon and PYCR1 have interaction. However, there is little information in detail on how mitochondrial Lon regulates metabolic reprogramming and metastasis through the interaction with PYCR1. In this study, it has been found that when Lon was overexpressed, the level of PYCR1 was elevated. Lon promotes collagen synthesis depends on PYCR1 upregulated and is proposed to makes the ECM stiffened which induces tumor metastasis. Furthermore, Both Lon and PYCR1 increase in NAD+/NADH ratio that may allow cells to transform to the Warburg effect. To sum up, this study demonstrated that the interaction of Lon-PYCR1 plays a crucial role in metabolic reprogramming and supports cancer cell proliferation and metastasis.

中文摘要 ..................................................................................... i
Abstract ..................................................................................... ii
Content ..................................................................................... iii
Chapter 1. Introduction ........................................................... 1
1.1 Cancer metabolism...........................................................................................1
1.2 Metastasis.........................................................................................................2
1.3 Lon protease.....................................................................................................4
1.4 Proline metabolism in cancer...........................................................................5
1.5 PYCR1 .............................................................................................................7
1.6 Collagen synthesis ...........................................................................................8
Chapter 2. Motivation and Rationale ..................................... 9
2.1 Motivation and Rationale.................................................................................9
Chapter 3. Material and methods ......................................... 10
3.1 Cell culture and treatment ..............................................................................10
3.2 Antibodies ......................................................................................................10
3.3 Cell lysate.......................................................................................................12
3.4 Plasmid...........................................................................................................13
3.5 Transfection....................................................................................................13
iv
3.6 Western blot ...................................................................................................14
3.7 Real time polymerase chain reaction (RT-PCR) ............................................15
3.8 NAD+/NADH assay .......................................................................................17
3.9 Wound Healing Assay and Migration Assay..................................................17
3.10 Immunofluorescence staining ......................................................................18
3.11 Adhesion assay .............................................................................................18
3.12 Statistical analysis ........................................................................................19
Chapter 4. Results................................................................... 20
4.1 Mitochondrial Lon induced the expression of PYCR1..................................20
4.2 Both Lon and PYCR1 increase the ratio of NAD+/NADH............................21
4.3 Overexpression of Lon and PYCR1 promotes collagen synthesis ................22
4.4 The collagen synthesis is promoted by Lon-induced PYCR1 .......................24
4.5 The up-regulated Lon or PYCR1 promote Epithelial–mesenchymal transition
(EMT) ..................................................................................................................25
4.6 PYCR1 and Lon are required for activation of EMT and metastasis ............26
4.7 Overexpression Lon and PYCR1 attenuates the adhesion ability of cancer
cells ......................................................................................................................27
Chapter 5. Conclusion ............................................................ 29
5.1 Conclusion .....................................................................................................29
v
Chapter 6. Discussion ............................................................. 30
6.1 The hypothetical model should do more experiments to confirm .................30
6.2 The collagen synthesis was not observed in the immunofluorescence staining
..............................................................................................................................31
6.3 The adhesion assay in the HSC3 cell line was not presented similar results.32
6.4 Highly expressed Lon were observed in PYCR1 overexpressed cancer cells
..............................................................................................................................32
6.5 The positive cycle for Lon-induced PYCR1 effect possibly promote kindlin 2
translocated into mitochondria.............................................................................33
Chapter 7. Figures .................................................................. 35
Figure 1. Identification of the expression of Lon and PYCR1 plasmid...............36
Figure 2. Mitochondrial Lon induced the expression of PYCR1 ........................39
Figure 3. Lon and PYCR1 increased the ratio of NAD+ and NADH and regulated
the redox homeostasis ..........................................................................................41
Figure 4. Kindlin2 is up-regulated by Lon overexpression .................................43
Figure 5. Overexpression of Lon and PYCR1 promotes collagen synthesis .......46
Figure 6. PYCR1 is involved in Lon-induced collagen synthesis .......................48
Figure 7. The up-regulated Lon and PYCR1 promote Epithelial–mesenchymal
transition (EMT) in cancer...................................................................................51
vi
Figure 8. The up-regulated Lon and PYCR1 promote matrix metalloproteinases
(MMPs) in cancer ................................................................................................55
Figure 9. The up-regulated Lon and PYCR1 promote migration of cancer cells 57
Figure 10. PYCR1 and Lon are required for activation of EMT and metastasis .60
Figure 11. The effect of Lon or PYCR1 on the adhesion ability of cancer cells .61
Figure 12. The hypothetical model ......................................................................63
Chapter8. References ............................................................. 64

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