臺灣博碩士論文加值系統

肺癌是全世界最常見的惡性腫瘤之一，並且位居癌症死亡原因之首。Gefitinib (Iressa, 艾瑞莎) 為表皮生長因子受體之酪氨酸激酶的抑制劑，在臨床上已有效使用在非小細胞肺癌患者的治療，然而於使用一段時間後會因產生抗藥性而失效，所以探討造成艾瑞莎產生抗藥性的分子機制是極為重要與迫切的。在本實驗中，我們發現在gefitinib-resistance的肺癌細胞株(PC9/GR)中FOXO3a的表現量低於gefitinib-sensitive的肺癌細胞株(PC9/WT)。因此我們在PC9/WT細胞中降低原本表現較高的FOXO3a，發現細胞因此對艾瑞莎產生抗藥性；相反的我們在PC9/GR表現FOXO3a，造成細胞對艾瑞莎具有敏感效果。在過去的研究指出，microRNA在許多癌症的發展當中扮演相當重要的角色，其中包含對藥物的抗藥性。故我們進一步探討microRNA是否會調控FOXO3a並導致艾瑞莎產生抗藥性。利用電腦網站之資料庫，預測出會標靶至FOXO3a的microRNA。接著我們利用即時定量聚合酶連鎖反應分析上述預測出的microRNA在PC9/WT與PC9/GR中的表現，並發現在PC9/GR中miR-155的表現高於PC9/WT。此外，我們在PC9/WT表現miR-155後，發現FOXO3a表現量下降並且增加細胞對艾瑞莎產生的抗藥性。總括而言，我們的研究指出miR-155與FOXO3a之間的相關性並證明miR-155和FOXO3a在對艾瑞莎產生抗藥的肺癌中可視為潛在的標靶治療方法。

Lung cancer is one of the most common malignancies worldwide. Epidermal growth factor receptor (EGFR) regulates many biological functions including proliferation, angiogenesis, anti-apoptosis and metastasis. A specific EGFR tyrosine kinase inhibitor, gefitinib (Iressa, ZD1839), has been developed to exhibit clinical efficacy in lung cancer patients. However, acquisition of resistance to gefitinib is also observed after treatment. It is critical and timely to understand the molecular mechanisms involved in gefitinib resistance. In this study, we found the expression level of FOXO3a was lower in gefitinib resistant PC9 (PC9/GR) cells than wild type PC9 (PC9/WT) cells. Kockdowned of FOXO3a in PC9/WT cells enhanced resistance of gefitinib ; consistantly, overexpression of FOXO3a make PC9/GR cells re-sensitize to gefitinib. MicroRNAs play critical role in multiple steps of cancer progression, including drug resistance. To define whether microRNAs involved in FOXO3a-mediated gefitinib-resistance, we predicted microRNAs which may target to FOXO3a by several softwares, such as miRWalk, DIANAmT , miRanda, miRDB and TargetScan. We further analyzed the expression of these candidate microRNAs in PC9/WT and PC9/GR cells by quantitative RT-PCR. We found that miR-155 was highly expressed in PC9/GR cells than in PC9/WT cells. Overexpression of miR-155 in PC9/WT cells downregulated FOXO3a and subsequently enhanced the resistance of gefitinib. Together, our study reveals a molecular link between miR-155 and FOXO3a and provides evidence that miR-155 and FOXO3a are potential therapeutic targets in gefitinib-resistant lung cancer.

目錄 (TABLE OF CONTENTS)
目錄.....................................................Ⅰ
致謝.....................................................Ⅲ
中文摘要.................................................Ⅴ
英文摘要.................................................Ⅵ
1. Introduction
1.1. Lung Cancer.........................................................................................1
1.2. EGFR Kinase Inhibitor........................................................................1
1.3. FOXO3a...............................................................................................2
1.4. microRNA and Cancer.........................................................................3
1.5. Hypothesis...........................................................................................4
2. Materials and Methods
2.1. Cell Line..............................................................................................5
2.2. Cell proliferation by MTT assay......................................................... 5
2.3. Gefitinib-induced cell death............................................................... 6
2.4. Western blotting..................................................................................6
2.5. RNA Isolation and Reverse Transcription..........................................7
2.6. Real-Time PCR Quantification...........................................................8
II
2.7. miRNA Overexpression and Inhibition..............................................9
2.8. Xenograft lung tumor growth assay...................................................10
2.9. Statistical assay..................................................................................10
3. Results
3.1. FOXO3a is critical for gefitinib resistance in lung cancer cells........11
3.2. Upregulation of miR-155 increased the gefitinib resistance by
down-regulation of FOXO3a....................................................................13
4. Discusion........................................................................................15
5. References.....................................................................................19
6. Figures.............................................................................................

1.Parkin, D. M., Bray, F., Ferlay, J., and Pisani, P. (2005) CA Cancer J Clin 55, 74-108
2.Faenza, I., Blalock, W., Bavelloni, A., Schoser, B., Fiume, R., Pacella, S., Piazzi, M., D''Angelo, A., and Cocco, L. (2012) FASEB J
3.Spira, A., and Ettinger, D. S. (2004) N Engl J Med 350, 379-392
4.Molina, J. R., Yang, P., Cassivi, S. D., Schild, S. E., and Adjei, A. A. (2008) Mayo Clin Proc 83, 584-594
5.Wheeler, D. L., Dunn, E. F., and Harari, P. M. (2010) Nat Rev Clin Oncol 7, 493-507
6.Muhsin, M., Graham, J., and Kirkpatrick, P. (2003) Nat Rev Drug Discov 2, 515-516
7.Sharma, S. V., Bell, D. W., Settleman, J., and Haber, D. A. (2007) Nat Rev Cancer 7, 169-181
8.Ethier, S. P. (2002) Semin Radiat Oncol 12, 3-10
9.Herbst, R. S., Fukuoka, M., and Baselga, J. (2004) Nat Rev Cancer 4, 956-965
10.Ranson, M., Hammond, L. A., Ferry, D., Kris, M., Tullo, A., Murray, P. I., Miller, V., Averbuch, S., Ochs, J., Morris, C., Feyereislova, A., Swaisland, H., and Rowinsky, E. K. (2002) J Clin Oncol 20, 2240-2250
11.Mitsudomi, T., and Yatabe, Y. (2007) Cancer Sci 98, 1817-1824
12.Inoue, A., Suzuki, T., Fukuhara, T., Maemondo, M., Kimura, Y., Morikawa, N., Watanabe, H., Saijo, Y., and Nukiwa, T. (2006) J Clin Oncol 24, 3340-3346
13.Inukai, M., Toyooka, S., Ito, S., Asano, H., Ichihara, S., Soh, J., Suehisa, H., Ouchida, M., Aoe, K., Aoe, M., Kiura, K., Shimizu, N., and Date, H. (2006) Cancer Res 66, 7854-7858
14.Kobayashi, S., Boggon, T. J., Dayaram, T., Janne, P. A., Kocher, O., Meyerson, M., Johnson, B. E., Eck, M. J., Tenen, D. G., and Halmos, B. (2005) N Engl J Med 352, 786-792
15.Pao, W., Miller, V. A., Politi, K. A., Riely, G. J., Somwar, R., Zakowski, M. F., Kris, M. G., and Varmus, H. (2005) PLoS Med 2, e73
16.Gazdar, A. F., and Minna, J. D. (2005) PLoS Med 2, e377
17.Arden, K. C. (2006) Exp Gerontol 41, 709-717
18.Greer, E. L., and Brunet, A. (2005) Oncogene 24, 7410-7425
19.Tsai, K. L., Sun, Y. J., Huang, C. Y., Yang, J. Y., Hung, M. C., and Hsiao, C. D. (2007) Nucleic Acids Res 35, 6984-6994
20.Hu, M. C., Lee, D. F., Xia, W., Golfman, L. S., Ou-Yang, F., Yang, J. Y., Zou, Y., Bao, S., Hanada, N., Saso, H., Kobayashi, R., and Hung, M. C. (2004) Cell 117, 225-237
21.Yang, J. Y., Zong, C. S., Xia, W., Yamaguchi, H., Ding, Q., Xie, X., Lang, J. Y., Lai, C. C., Chang, C. J., Huang, W. C., Huang, H., Kuo, H. P., Lee, D. F., Li, L. Y., Lien, H. C., Cheng, X., Chang, K. J., Hsiao, C. D., Tsai, F. J., Tsai, C. H., Sahin, A. A., Muller, W. J., Mills, G. B., Yu, D., Hortobagyi, G. N., and Hung, M. C. (2008) Nat Cell Biol 10, 138-148
22.Miyamoto, K., Araki, K. Y., Naka, K., Arai, F., Takubo, K., Yamazaki, S., Matsuoka, S., Miyamoto, T., Ito, K., Ohmura, M., Chen, C., Hosokawa, K., Nakauchi, H., Nakayama, K., Nakayama, K. I., Harada, M., Motoyama, N., Suda, T., and Hirao, A. (2007) Cell Stem Cell 1, 101-112
23.Tothova, Z., Kollipara, R., Huntly, B. J., Lee, B. H., Castrillon, D. H., Cullen, D. E., McDowell, E. P., Lazo-Kallanian, S., Williams, I. R., Sears, C., Armstrong, S. A., Passegue, E., DePinho, R. A., and Gilliland, D. G. (2007) Cell 128, 325-339
24.Myatt, S. S., and Lam, E. W. (2007) Nat Rev Cancer 7, 847-859
25.Slack, F. J., and Weidhaas, J. B. (2006) Future Oncol 2, 73-82
26.Kim, V. N., and Nam, J. W. (2006) Trends Genet 22, 165-173
27.Olsen, M. A., and Schechter, L. E. (1999) Gene 227, 63-69
28.Harfe, B. D. (2005) Curr Opin Genet Dev 15, 410-415
29.Gallardo, E., Navarro, A., Vinolas, N., Marrades, R. M., Diaz, T., Gel, B., Quera, A., Bandres, E., Garcia-Foncillas, J., Ramirez, J., and Monzo, M. (2009) Carcinogenesis 30, 1903-1909
30.Li, J., Huang, H., Sun, L., Yang, M., Pan, C., Chen, W., Wu, D., Lin, Z., Zeng, C., Yao, Y., Zhang, P., and Song, E. (2009) Clin Cancer Res 15, 3998-4008
31.Ryu, J. K., Hong, S. M., Karikari, C. A., Hruban, R. H., Goggins, M. G., and Maitra, A. (2010) Pancreatology 10, 66-73
32.Mukohara, T., Kudoh, S., Yamauchi, S., Kimura, T., Yoshimura, N., Kanazawa, H., Hirata, K., Wanibuchi, H., Fukushima, S., Inoue, K., and Yoshikawa, J. (2003) Lung Cancer 41, 123-130
33.Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., Barbisin, M., Xu, N. L., Mahuvakar, V. R., Andersen, M. R., Lao, K. Q., Livak, K. J., and Guegler, K. J. (2005) Nucleic Acids Res 33, e179
34.Simon, G., Ginsberg, R. J., and Ruckdeschel, J. C. (2001) Chest Surg Clin N Am 11, 165-188, ix
35.Ji, Y., Ma, S. L., Zhang, Y. P., Tang, J. J., Wu, Y. M., and Lu, Y. J. (2009) Anticancer Drugs 20, 832-837
36.Zhou, X., Zheng, M., Chen, F., Zhu, Y., Yong, W., Lin, H., Sun, Y., and Han, X. (2009) Anat Rec (Hoboken) 292, 1122-1127
37.Ma, B. B., Lui, V. W., Poon, F. F., Wong, S. C., To, K. F., Wong, E., Chen, H., Lo, K. W., Tao, Q., Chan, A. T., Ng, M. H., and Cheng, S. H. (2010) Invest New Drugs 28, 326-333