肺癌在全世界位居所有癌症中致死率最高之疾病。我們最近研究結果發現在肝癌病人腫瘤中，人類的核糖-5-磷酸異構酶A (RPIA)會過量表現，且RPIA的表現量可調控腫瘤的生成。然而RPIA在肺癌中所扮演的角色並不清楚。本篇論文中，我發現抑制RPIA表現量會導致肺癌細胞生長減緩，並誘發自噬作用與細胞凋亡以及細胞衰老作用產生。
首先，我們在肺癌病人癌化組織切片中發現相對於周邊較正常組織相比，其RPIA有過度表現的現象。因此，我們利用慢病毒感染方式感染於A549肺癌細胞中，分別建立抑制RPIA表現的兩株獨立細胞株；在這兩株獨立細胞株中，發現細胞型態會產生自噬泡，且會增加acridine orange 呈色，並導致自噬體的堆積，產生GFP-LC3 的小點;而在分子機轉中，發現這兩株RPIA抑制表現的細胞株中，會促使LC3-Ⅱ增加與p62的減少，這些現象證明在A549細胞抑制RPIA會活化自噬作用的產生。另外，發現在A549抑制RPIA表現，會造成Bax、cleaved PARP、caspase 3蛋白質表現量增加，這些說明抑制RPIA表現會活化粒線體所誘導的細胞凋亡機制。再者，在肺癌細胞中抑制RPIA會引發細胞衰老且增加p21的表現。最重要的是在肺癌細胞中抑制RPIA會導致活性氧化物(Reactive Oxygen Species)的增加；當在RPIA抑制的A549細胞中，加入ROS的清除劑N-乙酰基-L-半胱氨酸(NAC)，會降低ROS的量，並且可逆轉由於抑制RPIA所造成的自噬作用、細胞凋亡與細胞衰老現象的分子蛋白的表現情形。
我們的研究發現在A549肺癌細胞中抑制RPIA表現，會誘導活性氧物種的增加，並導致自噬作用、細胞凋亡、細胞衰老之作用產生。我們的研究闡明抑制RPIA表現量可作為一個新興治療肺癌的策略。

Lung cancer is a leading cause of cancer death worldwide. Our recent study showed that human ribose-5-phosphate isomerase A (RPIA) regulates tumorigenesis in liver cancer. However, the role of RPIA in lung cancer is not known. Here we report that knockdown of RPIA diminishes lung cancer proliferation by the activation of autophagy, apoptosis, and cellular senescence.
First, we detected RPIA up-regulation in the human lung cancer versus adjust normal tissue by IHC using tissue array. Therefore we established two independent lentiviral shRPIA knockdown cell lines in A549 lung cancer cells. Both RPIA knockdown lung cancer cell lines in cellular level displayed autophagic vacuoles, enhanced acridine orange staining, GFP-LC3 punctae, and accumulated autophagosomes and in molecular level both showed elevated levels of LC3-Ⅱ and reduced levels of p62, which together suggests RPIA knockdown activates autophagy in A549 cells. In addition, RPIA knockdown in A549 cells induced mitochondria mediated apoptosis by exhibiting increased levels of Bax, cleaved PARP and caspase-3 and elevated levels of apoptotic cells. Moreover, RPIA knockdown triggered cellular senescence and increased levels of p21 in A549 cells. Importantly, RPIA knockdown increased reactive oxygen species (ROS) levels in A549 cells. Treatment of ROS scavenger N-acetyl-L-cysteine (NAC) reverts the activation of autophagy ,apoptosis and cellular senescence by RPIA knockdown in A549 cells. In conclusion, RPIA knockdown induces oxidative stress to activate autophagy, apoptosis, and cellular senescence in lung cancer cells. Our study sheds new light on RPIA suppression in lung cancer therapy.

Table of contents
Abstract II
中文摘要 III
誌謝……………..…………………………………………………………………….V
Table of contents…………………………………………………………………... VII
Chapter 1 Introduction 1
1-1 Lung cancer 1
1-2 Lung cancer, aging, and metabolism 1
1-3 Programmed cell death pathways in lung cancer 2
1-3-1 Apoptosis 2
1-3-2 Autophagy 3
1-3-3 Cellular senescence 4
1-4 Lung cancer and ROS 5
1-5 Aims 6
Chapter 2 Materials and Methods 7
2-1 DNA plasmid generation 7
2-2 Cell culture 7
2-3 Immunohistochemistry 8
2-4 Lentiviral Infection 8
2-5 RNA extraction and Quantitative Real-time PCR 9
2-6 Protein extraction and Western blot 10
2-7 Colony formation assay 11
2-8 Water-Soluble Tetrazolium Salt-1 (WST-1) 11
2-9 Transmission electron microscopy (TEM) 12
2-10 Acridine orange staining 12
2-11 Apoptosis cells detection 12
2-12 Senescence assay 13
2-13 Reactive Oxygen Species (ROS) cells detection 13
2-14 Statistical analysis 14
Chapter 3 Results 15
3-1 Increased RPIA expression was detected in the tumor biopsy of lung adenocarcinoma patients 15
3-2 Establishment of lentiviral-shRNA RPIA knockdown cell lines in A549 lung cancer cells 15
3-3 Knockdown of RPIA decreased the oncogenicity in A549 cells. 16
3-4 Knockdown of RPIA induced mitochondria-mediated apoptosis in A549 cells. 17
3-5 Knockdown of RPIA induces autophagy in A549 lung cancer cells 18
3-6 Inhibition of autophagy by autophagy inhibitors 3-MA and CQ enhances apoptosis induced by shRPIA in A549 cells. 19
3-7 Knockdown of RPIA triggered cellular senescence and enhanced p21 expression levels in A549 cells 19
3-8 Reactive oxygen species induced by shRPIA caused autophagy and apoptosis in A549 cells 20
Chapter 4 Discussion 22

Table 1. Q-PCR primer 26
Table 2. Antibodies list 26
Figure 1. Increased RPIA expression is detected in the tumor biopsy of lung adenocarcinoma patients. 27
Figure 2. The mRNA and protein expression levels of RPIA were reduced in the RPIA shRNA knockdown A549 lung cancer cells. 29
Figure 3. Knockdown of RPIA decreased the oncogenicity of A549 cells. 31
Figure 4. Knockdown of RPIA induced mitochondria mediated apoptosis in A549 cells. 33
Figure 5. Knockdown of RPIA induced autophagosome formation, reduced p62 and increased LC3-Ⅱ protein levels in A549 cells. 35
Figure 6. The appearance of autophagosome formation in shRPIA knockdown A549 cells by transmission electron microscopy. 38
Figure 7. Knockdown of RPIA triggered cell senescence and enhanced p21 expression levels in A549 cells. 40
Figure 8. The increased levels of reactive oxygen species induced by shRPIA knockdown mediate the expression of autophagy, apoptosis and cellular senescence related proteins in the A549 cells. 42
Figure 9. Summary 44
Appendix…………………………………………………………………………….45
References………... …..49

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