臺灣博碩士論文加值系統

近幾十年來，肺癌在世界與台灣是癌症死亡率的主要原因。自噬作用是一種細胞內的防禦機制，藉由分解不正常的物質、胞器及外來病原體以維持細胞內的平衡狀態。自噬作用機制並不是毫無選擇性的降解途徑。不同的物質會透過一些特定的自噬作用受體進行辨識後再進行降解。其中，SQSTM1是一個重要的選擇性自噬作用受體，能使泛素化的物質接上LC3形成自噬體，接著進行降解作用。然而SQSTM1在肺癌細胞中扮演的角色以及能否作為治療的標靶仍不清楚。在本研究中，我們利用CRISPR/Cas9基因編輯技術將肺癌細胞中的SQSTM1基因剔除，並以西方墨點法及免疫螢光染色驗證這些細胞確實無法表現SQSTM1蛋白。此外，我們進一步以核酸定序找出被剔除的SQSTM1基因序列片段，利用CRISPR/Cas9基因編輯技術確實能將SQSTM1剔除而不會影響其他選擇性自噬作用受體OPTN和NDP52及自噬路徑的關鍵蛋白LC3，但剔除SQSTM1基因的肺癌細胞，會造成細胞型態的改變，以及降低細胞的移行能力，RhoA與Cdc42的蛋白表現在SQSTM1基因剔除的肺癌細胞中也會大幅下降，但不會影響Rac1的表現。再者，剔除SQSTM1基因會降低自噬作用抑制藥物Chloroquine在肺癌細胞中的藥效，而自噬作用促進藥物Rapamycin的藥效則不受到影響。總結上述結果，我們證實在肺癌細胞中剔除SQSTM1基因會影響細胞的型態、移行能力及特定抗癌化合物的藥效，我們推測SQSTM1是具有潛力的選擇性自噬作用受體，可開發作為未來肺癌的治療標靶。

Lung cancer is one of leading cause of cancer death in the world and Taiwan during the past few decades. Autophagy is a mechanism of the cells that disassembles aberrant components, organelles, and intracellular pathogens to maintain the cellular homeostasis. The process of autophagy is not simply a non-selective degradation pathway. Selective autophagy receptors have been identified which specifically mediate the selective autophagosomal degradation of a variety of cargoes. SQSTM1 is a pivotal selective autophagy receptor recruits LC3-containing autophagosomes to ubiquitinated cargos for degradation. However, the role of SQSTM1 in lung cancer cells and whether can be applied as a target for lung cancer therapy are still unclear yet. In this study, we generated the SQSTM1 knockout in lung cancer cells using the CRISPR/Cas9 gene editing technique. The SQSTM1 knockout cells were verified by western blot and immunofluorescence staining. Furthermore, the SQSTM1 knockout fragments were determined by DNA sequencing. The results showed that CRISPR/Cas9 gene editing specifically knockout SQSTM1 but not delete other autophagy receptors, such as OPTN and NDP52, and autophagy pathway key protein LC3. Nonetheless, we found that knockout of SQSTM1 induced the morphological alteration and attenuated the cell migration ability in the lung cancer cells. The protein levels of RhoA and Cdc42 but not Rac1 were dramatically decreased in both the SQSTM1 knockout lung cancer cells. Besides, knockout of SQSTM1 reduced the efficacy of Chloroquine, an autophagy inhibitor, in lung cancer cells; however, it did not alter the efficacy of Rapamycin, an autophagy inducer. Taken together, this study demonstrated that knockout of SQSTM1 affected the cellular morphology, migration and specific anticancer compound efficacy in the lung cancer cells. We suggest that SQSTM1 is a potential selective autophagy receptor that can be developed as target for lung cancer therapy.

Contents i
中文摘要 iii
Abstract iv
Abbreviations v
1. Introduction 1
1.1. Lung cancer 1
1.2. Biological functions of autophagy pathway 1
1.3. Selective autophagy receptor 2
1.4. Selective autophagy receptors in cancer progression 2
1.5. Rho GTPases in cancer metastasis 3
1.6. Autophagy drugs for cancer therapy 3
1.7. CRISPR/Cas9 knockout system 4
1.8. Purpose of this study 5
2. Materials and methods 6
2.1. Chemicals and reagents 6
2.2. Antibodies 6
2.3. Cell lines and cell culture 7
2.4. Western blot analysis 7
2.5. Immunofluorescence staining and confocal microscopy 8
2.6. Cell growth assay 9
2.7. Reverse transcription-polymerase chain reaction (RT-PCR) 9
2.8. Generation of knockout cell lines using CRISPR/Cas9 gene editing technique 10
2.9. Cell viability assay 10
2.10. Wound-healing assay 11
2.11. Statistical analysis 11
3. Results 12
3.1. Generation and identification of SQSTM1 knockout in A549 lung cancer cells 12
3.2. Generation and identification of SQSTM1 knockout in H1975 lung cancer cells …………………………………………………………………………………..13
3.3. The protein expressions of autophagy pathway in the SQSTM1 knockout lung cancer cells 13
3.4. The cellular morphology and migration ability in the SQSTM1 knockout lung cancer cells 14
3.5. The efficacy of anticancer compounds in the SQSTM1 knockout lung cancer cells.......... 15
4. Conclusions 15
5. References 16
6. Figures 22
Figure 1. The generation of SQSTM1 gene knockout in lung cancer cells using CRISPR/Cas9 gene editing. 22
Figure 2. The verification of protein levels of SQSTM1 in the A549 SQSTM1 knockout cells by western blot and confocal microscopy. 23
Figure 3. The DNA sequencing of the SQSTM1 knockout in the A549 cells. 24
Figure 4. The deletion of 138 nucleotides in the A549 SQSTM1 KO #4 cells. 25
Figure 5. The generation of SQSTM1 gene knockout in lung cancer cells using CRISPR/Cas9 gene editing. 26
Figure 6. The verification of protein levels of SQSTM1 in the H1975 SQSTM1 knockout cells by western blot and confocal microscopy. 27
Figure 7. The deletions of 372 and 204 nucleotides in the H1975 SQSTM1 KO #5 and #6 cells. 28
Figure 8. The protein alteration of autophagy pathways in the SQSTM1 knockout cells of A549 and H1975. 29
Figure 9. The morphology of A549 SQSTM1 knockout cells differs from A549 wild-type cells. 30
Figure 10. The morphology of H1975 SQSTM1 knockout cells differs from H1975 wild-type cells. 31
Figure 11. Comparison of cell growth ability in the A549, H1975 and SQSTM1 knockout cells. 32
Figure 12. Knockout of SQSTM1 attenuates the cell migration ability in A549 cells. 33
Figure 13. Knockout of SQSTM1 decreases Rho GTPase family protein expression in A549 and H1975 cells 34
Figure 14. Knockout of SQSTM1 reduces the efficacy of Chloroquine. 35
Figure 15. Knockout of SQSTM1 does not reduce the efficacy of Rapamycin. 36
Figure 16. The model of SQSTM1 knockout in lung cancer cells. 37
7. Appendixes 38
Appendix 1. The major types of lung cancer. 38
Appendix 2. Selective autophagy. 39
Appendix 3. Domain architecture of the known characterized autophagy receptors. 40
Appendix 4. Roles of Cdc42, Rac and Rho in cellular migration. 41
Appendix 5. CRISPR/Cas9 system. 42

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