臺灣博碩士論文加值系統

粒線體DNA跟癌細胞的能量供應及代謝息息相關，先前有研究發現肺癌在腫瘤生長形成及惡化的過程中，粒線體DNA會出現愈來愈明顯的減少情形，或許可以說明粒線體DNA減少對細胞造成的影響不僅是能量供應的減少，可能還有其他有待探討的影響。
為了瞭解粒線體DNA拷貝數改變對肺癌細胞的代謝造成的影響，我們觀察了21組非小細胞肺癌病人的腫瘤組織及同器官之正常組織的粒線體DNA，藉由減少及增加TFAM基因的表現來讓肺癌細胞株的粒線體DNA增加或減少，接著用海馬生物能量代謝測定儀去測量肺癌細胞的耗氧率及乳酸產生率，並觀察其生長速度、爬行能力、侵犯能力以及施加抗癌藥物IRESSA後存活率的變化(抗藥性)，另外，我們還觀察了一些既可以促進糖解作用又可以參與在合成脂質、蛋白質及核酸等大分子物質過程中的代謝途徑的表現量及活化程度，包括PI3K/Akt/mTORC及Myc為主的相關代謝途徑，並藉由施加PI3K/Akt途徑的抑制劑LY294002，同樣觀察這些代謝途徑對肺癌細胞生長速度、爬行能力、侵犯能力及抗藥性的影響。
我們的結果發現，相較於正常組織而言，粒線體DNA在體積較小的病人腫瘤中偏向增加的情形，但在體積較大、發展到較晚的病人腫瘤中則偏向減少的趨勢，而在細胞株的部分，結果顯示粒線體DNA減少會使得肺癌細胞的有氧呼吸效率、生長速度及對IRESSA的抗藥性都降低，但會促進糖解作用及PI3K/Akt/mTORC及Myc為主的合成代謝途徑的表現及活化，這可能會對不同種類的細胞株的爬行及侵犯能力造成不同程度的影響，也或許這就是粒線體DNA在體積大的腫瘤中傾向於減少的原因。相較之下，粒線體DNA增加則似乎只會藉由促進肺癌細胞的有氧呼吸及能量供應來促進癌細胞的生長速度及對IRESSA的抗藥性，對於糖解作用及PI3K/Akt/mTORC及Myc為主的合成代謝途徑則並無明顯影響。我們的研究結果或許有助於釐清粒線體DNA在肺癌細胞裡扮演的角色及造成的影響，並能為未來針對抗癌療法或藥物的研究提供一個新的標靶代謝途徑。

Copy number of human mitochondrial DNA (mtDNA) is closely associated with cancer energy metabolism. However, it was found that mtDNA copy number reduced in the process of tumorigenesis and worsening, which suggests that mtDNA copy number reduction leads to not only the energy supply change but also some other unclear effects.
In order to further investigate the role of mtDNA copy number alteration in cancer cells, we observed the mtDNA copy number change of 21 pairs of tumor part and non-tumor part of the same organ from 21 NSCLC patients, and used human non-small cell lung cancer (NSCLC) cell lines as study materials, and knock-down and overexpressed gene TFAM of NSCLC cell lines to reduce the mtDNA copy number, and then used Seahorse XFe24 Extracellular Flux Analyzer to measure the change of oxygen consumption rates and lactate production rates. Furthermore, we evaluated its proliferation rates, and observed the migration and invasion propensity change with wound-healing and transwell invasion assay, and treated cancer cells with anticancer drug Iressa to evaluate its drug resistance. In addition, we also examined the expression levels and activation levels of glycolysis and its related regulation pathways, like the PI3K/Akt/mTORC signaling pathway and Myc-associated pathway, and treated cancer cells with PI3K/Akt pathway inhibitor LY294002 to evaluate its metabolic effects on cell proliferation, drug resistance, and migration and invasion propensity.
Our results from primary samples of NSCLC patients revealed that mtDNA copy number tended to increase in tumor with smaller size, but tended to reduce in tumor with larger size, or in the late phase of tumor progression, compared to non-tumor part. The results from NSCLC cell lines revealed that reduced mtDNA copy number could reduce aerobic respiration and thus inhibit the cell proliferation and drug resistance, and also promote the glycolytic catabolism pathway and Akt-dependent and Myc-dependent anabolism pathway, which might lead to different level of effects on the migration and invasion propensity of different cell lines and might be the reason why mtDNA copy number tended to reduce in tumor with larger size. On the contrary, mtDNA copy number increase promoted aerobic respiration and energy supply and thus promoted the cell proliferation and drug resistance, but didn’t change the glycolytic catabolism pathway and Akt-dependent and Myc-dependent anabolism pathway. Our findings helped to clarify the role of mtDNA copy number alteration in human NSCLC and provided a novel and potential drug target pathway for cancer researches in the future.

Chinese Abstract-----------------------------------------------------------------i
English Abstract----------------------------------------------------------------iii
Contents--------------------------------------------------------------------------v
Introduction----------------------------------------------------------------------1
I. Mitochondrial DNA------------------------------------------------------- 1
II. The relationship between mtDNA copy number and tumorigenesis and metastasis--------------------------------------------------------------------1
III. The role of mitochondrial transcription factor A (TFAM) in maintaining mtDNA copy number---------------------------------------------2
IV. Metabolic reprogramming-----------------------------------------------3
V. Specific aim-----------------------------------------------------------------5
Materials and Methods-----------------------------------------------------------7
I. Materials----------------------------------------------------------------------7
II. Methods----------------------------------------------------------------------7
1. Collection of patient tissues and DNA extraction--------------------7
2. TFAM knockdown with lentivirus infection--------------------------8
3. Western blot analysis----------------------------------------------------8
4. Relative mtDNA copy number evaluation----------------------------9
5. Oxygen consumption rate measurement------------------------------9
6. Lactate production rate measurement---------------------------------9
7. Migration and invasion assay-----------------------------------------10
8. Drug resistance to IRESSA--------------------------------------------10
9. Statistical Analysis------------------------------------------------------10

Results--------------------------------------------------------------------------11
1. mtDNA copy number tends to decline in the process of tumor progression---------------------------------------------------------------------11
2. Comparison of TFAM expression level and relative mtDNA copy number of five non-small cell lung cancer (NSCLC) cell lines---------12
3. Comparison of migration and invasion propensity of five non-small cell lung cancer (NSCLC) cell lines----------------------------------------12
4. Comparison of drug resistance of five non-small cell lung cancer (NSCLC) cell lines to anticancer drug IRESSA--------------------------13
5. TFAM knock-down leads to mtDNA copy number decline, electron transport chain function, and aerobic respiration rate--------------------14
6. mtDNA copy number decline upregulated glycolysis-----------------16
7. mtDNA copy number decline upregulated Akt-dependent and Myc -dependent biosynthetic pathway--------------------------------------------16
8. mtDNA copy number decline have different effects on the EMT levels, migration activity and invasion propensity of different cell lines-------18
9. mtDNA copy number decline inhibited NSCLC cell proliferation---19
10. mtDNA copy number decline reduced the drug resistance to IRESSA-------------------------------------------------------------------------19
11. Blocking PI3K/Akt/mTORC pathway reduced the migration activity of NSCLC cells after mtDNA copy number decline----------------------20
12. Blocking PI3K/Akt/mTORC pathway reduced the invasion propensity after mtDNA copy number decline -----------------------------------------20
13. Blocking PI3K/Akt/mTORC pathway inhibited NSCLC cell proliferation after mtDNA copy number decline--------------------------21
14. Blocking PI3K/Akt/mTORC pathway inhibited the drug resistance of NSCLC cells to IRESSA after mtDNA copy number decline-----------21
15. TFAM overexpression leads to the increase of mtDNA copy number, electron transport chain function, and aerobic respiration rate----------21
16. mtDNA copy number increase had no effect on glycolysis---------23
17. mtDNA copy number increase had no effect on Akt-dependent and Myc-dependent pathway-----------------------------------------------------24
18. mtDNA copy number increase promotes EMT levels, migration and invasion propensity-----------------------------------------------------------25
19. mtDNA copy number decline inhibited NSCLC cell proliferation--------------------------------------------------------------------26
20. mtDNA copy number decline inhibited the drug resistance to IRESSA-------------------------------------------------------------------------26
Discussion----------------------------------------------------------------------28
References----------------------------------------------------------------------34
Figures--------------------------------------------------------------------------37
Tables--------------------------------------------------------------------------100

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