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博碩士論文 etd-0801117-090643 詳細資訊
Title page for etd-0801117-090643
論文名稱
Title
探討GREM1 基因對BFTC905 之細胞週期調控
Studies on the cell cycle regulation roles of GREM1 gene in BFTC905 cells
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
54
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2017-08-28
繳交日期
Date of Submission
2017-09-01
關鍵字
Keywords
膀胱尿路上皮細胞癌、GREM1、細胞週期、CKI、抑癌基因
Urinary bladder urothelial carcinoma, Tumor suppressor, GREM1, Cell cycle, CKI
統計
Statistics
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中文摘要
膀胱尿路上皮細胞癌 (Urinary Bladder Urothelial Carcinoma, UBUC) 是泌尿系統中的第二大癌症,不易治療且復發性高,故治療耗費很大的時間和金錢成本。危險因子像是工作環境的暴露及疾病的誘發膀胱癌。近年來因為全基因組掃描的普遍化,發現膀胱癌和基因的異常有關。初步的資料探勘 (data mining) 發現GREM1在可能參與癌症相關的訊號路徑。GREM1在膀胱癌中極少被研究,故探討GREM1與膀胱癌細胞增生之關係。在細胞內大量表現GREM1基因,發現會造成細胞週期G0/G1的停滯,增加調控細胞週期CKIs的蛋白表現,抑制細胞遷移能力。相反的將高內生性的BFTC905膀胱癌細胞株,抑制GREM1的基因表現,反而使細胞週期S phase增加,促進細胞增生,降低調控細胞週期CKIs蛋白質的表現、增加非貼覆性生長能力、細胞遷移和侵犯能力。GREM1在膀胱癌細胞中促進週期G0/G1停滯,抑制細胞增生,具有腫瘤抑制基因的功能。
Abstract
Urinary bladder urothelial carcinoma (UBUC) is the second largest cancer in urine system. It is hard to cure and highly recurrent so it costs lots of time and money to completely cure. Risk factor of UBUC includes occupational exposure and disease inducing. In recent years, human whole genome screening is universal. There is a discovery of relation between abnormal gene and bladder cancer due to the generality of data mining that GREM1 has abnormally high expression in cells of patients which suffer from UBUC. GREM1 is rarely studied in bladder cancer. In this study, we explored the relation between GREM1 and cell proliferation of UBUC. Overexpression of GREM1 in cells induced G0/G1 cell cycle arrest, increased CKIs protein expression of control cell cycle, and downregulated cell migration. In contrast, when downregulation of GREM1 in BFTC905 bladder cancer cells, it accelerated cell cycle S phase, cell proliferation, anchorage-independent cell growth, cell migration, invasion, and inhibited CKIs protein expression of control cell cycle cell. Therefore, our data indicated that GREM1 functions as a tumor suppressor in bladder cancer by inducing G0/G1 cycle arrest and downregulating cell growth.
目次 Table of Contents
論文審定書 i
誌謝 ii
中文摘要 iii
英文摘要 iv
目錄 v
圖次 viii
表次 ix
英文縮寫表 x
壹、 緒論 (Introduction) 1
一、 簡介膀胱癌 1
二、 簡介Gremlin 1 (GREM1) 2
三、 TGFB superfamily 4
四、 Cell cycle 4
貳、 實驗材料與方法 (Materials and methods) 5
一、 細胞培養 5
1. 膀胱癌細胞株 5
2. 解凍細胞 6
3. 細胞繼代培養 6
4. 冷凍保存細胞 6
二、質體製備 7
1. 質體 7
2. 質體轉型 7
3. 瓊脂膠體電泳 7
三、基因轉殖 9
1. 質體轉染 9
2. 慢病毒感染 9
四、cDNA 製備與Quantitative real-time PCR 9
1. Total RNA 萃取 9
2. Quantitative real-time PCR 10
五、蛋白質電泳與西方墨點法 10
1. 蛋白質純化 10
2. 西方墨點法 10
六、 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 11
七、 5-bromo-2-deoxyuridine (BrdU) assay 11
八、 細胞週期分析 11
九、 細胞遷移性試驗 12
1. Wound healing assay 12
十、Transwell assay 12
1. Invasion assay 12
2. Migration assay 13
十一、 細胞轉型分析 (Soft agar assay) 13
十二、 實驗數據統計分析 14
參、 結果 (Results) 24
一、 分析人類膀胱癌細胞株之內生性GREM1的mRNA與蛋白質表現 24
二、 高度表現GREM1使細胞停滯在G0/G1期,抑制細胞遷移能力 24
三、 在BFTC905細胞穩定抑制GREM1表現,促進cell cycle progression、細胞增生及非貼覆性生長之能力 25
四、在BFTC905株抑制GREM1的表現,促進細胞遷移 (Migration)及侵犯(Invasion) 25
五、 在BFTC905細胞穩定抑制GREM1表現,使調控G1/S CKIs蛋白質表現降低 26
肆、 討論 (Discussion) 38
伍、 參考文獻 (References) 39
參考文獻 References
Adams, P. D., and W. G. Kaelin. 1995. Transcriptional control by E2F. In: Seminars in cancer biology. p 99-108.
Avsian-Kretchmer, O., and A. J. Hsueh. 2004. Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists. Molecular endocrinology 18(1):1-12.
Brazil, D. P., R. H. Church, S. Surae, C. Godson, and F. Martin. 2015. BMP signalling: agony and antagony in the family. Trends in cell biology 25(5):249-264.
Chan, K. S., I. Espinosa, M. Chao, D. Wong, L. Ailles, M. Diehn, H. Gill, J. Presti, H. Y. Chang, and M. van de Rijn. 2009. Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells. Proceedings of the National Academy of Sciences 106(33):14016-14021.
Church, R. H., A. Krishnakumar, A. Urbanek, S. Geschwindner, J. Meneely, A. Bianchi, B. Basta, S. Monaghan, C. Elliot, and M. Strömstedt. 2015. Gremlin1 preferentially binds to bone morphogenetic protein-2 (BMP-2) and BMP-4 over BMP-7. Biochemical Journal 466(1):55-68.
Davis, H., S. Irshad, M. Bansal, H. Rafferty, T. Boitsova, C. Bardella, E. Jaeger, A. Lewis, L. Freeman-Mills, and F. C. Giner. 2015. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche. Nature medicine 21(1):62-70.
Derynck, R., and K. Miyazono. 2008. The TGF-[beta] family. CSHL Press.
Donovan, J., and J. Slingerland. 2000. Transforming growth factor-β and breast cancer: cell cycle arrest by transforming growth factor-β and its disruption in cancer. Breast Cancer Research 2(2):116.
Edge, S., D. Byrd, C. Compton, A. Fritz, F. Greene, and A. Trotti. 2010. Urinary bladder. AJCC cancer staging manual 7
Ehrlich, M., O. Gutman, P. Knaus, and Y. I. Henis. 2012. Oligomeric interactions of TGF‐β and BMP receptors. FEBS letters 586(14):1885-1896.
Elangovan, S., T.-C. Hsieh, and J. M. Wu. 2008. Growth inhibition of human Mda-Mb-231 breast cancer cells by Δ-tocotrienol is associated with loss of cyclin D1/Cdk4 expression and accompanying changes in the state of phosphorylation of the retinoblastoma tumor suppressor gene product. Anticancer research 28(5A):2641-2647.
Fine, S. W., P. A. Humphrey, L. P. Dehner, M. B. Amin, and J. I. Epstein. 2005. Urothelial neoplasms in patients 20 years or younger: a clinicopathological analysis using the world health organization 2004 bladder consensus classification. The Journal of urology 174(5):1976-1980.
Gandini, S., E. Botteri, S. Iodice, M. Boniol, A. B. Lowenfels, P. Maisonneuve, and P. Boyle. 2008. Tobacco smoking and cancer: A meta‐analysis. International journal of cancer 122(1):155-164.
Gatza, C. E., S. Y. Oh, and G. C. Blobe. 2010. Roles for the type III TGF-β receptor in human cancer. Cellular signalling 22(8):1163-1174.
Giacinti, C., and A. Giordano. 2006. RB and cell cycle progression. Oncogene 25(38):5220.
Gubern, A., M. Joaquin, M. Marquès, P. Maseres, J. Garcia-Garcia, R. Amat, D. González-Nuñez, B. Oliva, F. X. Real, and E. de Nadal. 2016. The N-Terminal Phosphorylation of RB by p38 Bypasses Its Inactivation by CDKs and Prevents Proliferation in Cancer Cells. Molecular cell 64(1):25-36.
Hakenberg, O. W. 2010. Urinary Diversion After Radical Cystectomy for Muscle-Invasive Bladder Cancer. European Urology Supplements 9(10):735. doi: http://dx.doi.org/10.1016/j.eursup.2010.09.004
Hengst, L., V. Dulic, J. M. Slingerland, E. Lees, and S. I. Reed. 1994. A cell cycle-regulated inhibitor of cyclin-dependent kinases. Proceedings of the National Academy of Sciences 91(12):5291-5295.
Hoffman, A. M., and P. Cairns. 2011. Epigenetics of kidney cancer and bladder cancer. Epigenomics 3(1):19-34.
Hogan, B. 1996. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes & development 10(13):1580-1594.
Hsu, D. R., A. N. Economides, X. Wang, P. M. Eimon, and R. M. Harland. 1998. The Xenopus Dorsalizing Factor Gremlin Identifies a Novel Family of Secreted Proteins that Antagonize BMP Activities. Molecular Cell 5(1):673-683.
Kawakami, Y., T. Ishikawa, M. Shimabara, N. Tanda, M. Enomoto-Iwamoto, M. Iwamoto, T. Kuwana, A. Ueki, S. Noji, and T. Nohno. 1996. BMP signaling during bone pattern determination in the developing limb. Development 122(11):3557-3566.
Khokha, M. K., D. Hsu, L. J. Brunet, M. S. Dionne, and R. M. Harland. 2003. Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning. Nature genetics 34(3):303-307. doi: 10.1038/ng1178
Kim, M., S. Yoon, S. Lee, S. A. Ha, H. K. Kim, J. W. Kim, and J. Chung. 2012. Gremlin-1 induces BMP-independent tumor cell proliferation, migration, and invasion. PLoS One 7(4):e35100.
Larré, S., J. W. Catto, M. S. Cookson, E. M. Messing, S. F. Shariat, M. S. Soloway, R. S. Svatek, Y. Lotan, A. R. Zlotta, and H. B. Grossman. 2013. Screening for bladder cancer: rationale, limitations, whom to target, and perspectives. European urology 63(6):1049-1058.
Laurila, R., S. Parkkila, J. Isola, A. Kallioniemi, and E.-L. Alarmo. 2013. The expression patterns of gremlin 1 and noggin in normal adult and tumor tissues. International Journal of Clinical and Experimental Pathology 6(7):1400.
Lin, H. Y., X.-F. Wang, E. Ng-Eaton, R. A. Weinberg, and H. F. Lodish. 1992. Expression cloning of the TGF-β type II receptor, a functional transmembrane serine/threonine kinase. Cell 68(4):775-785.
Lopez-Beltran, A., and R. Montironi. 2004. Non-invasive urothelial neoplasms: according to the most recent WHO classification. European urology 46(2):170-176.
Ma, B., Q. Kang, L. Qin, L. Cui, and C. Pei. 2014. TGF-β2 induces transdifferentiation and fibrosis in human lens epithelial cells via regulating gremlin and CTGF. Biochemical and biophysical research communications 447(4):689-695.
Meng, X.-M., A. C. Chung, and H. Y. Lan. 2013. Role of the TGF-β/BMP-7/Smad pathways in renal diseases. Clinical science 124(4):243-254.
Merino, R., J. Rodriguez-Leon, D. Macias, Y. Ganan, A. Economides, and J. Hurle. 1999. The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb. Development 126(23):5515-5522.
Michos, O., L. Panman, K. Vintersten, K. Beier, R. Zeller, and A. Zuniga. 2004. Gremlin-mediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis. Development 131(14):3401-3410.
Mitola, S., C. Ravelli, E. Moroni, V. Salvi, D. Leali, K. Ballmer-Hofer, L. Zammataro, and M. Presta. 2010. Gremlin is a novel agonist of the major proangiogenic receptor VEGFR2. Blood 116(18):3677-3680.
Morgan, D. O. 1995. Principles of CDK regulation. Nature 374(6518):131-134.
Morikawa, M., R. Derynck, and K. Miyazono. 2016. TGF-β and the TGF-β family: context-dependent roles in cell and tissue physiology. Cold Spring Harbor perspectives in biology 8(5):a021873.
Pang, K. H., and J. W. Catto. 2013. Bladder cancer. Surgery (Oxford) 31(10):523-529.
Pearce, J. J., G. Penny, and J. Rossant. 1999. A mouse cerberus/Dan-related gene family. Developmental biology 209(1):98-110.
Polyak, K., J. Kato, M. J. Solomon, C. J. Sherr, J. Massague, J. M. Roberts, and A. Koff. 1994. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes & development 8(1):9-22.
Rodrigues-Diez, R., R. R. Rodrigues-Diez, C. Lavoz, G. Carvajal, A. Droguett, A. B. Garcia-Redondo, I. Rodriguez, A. Ortiz, J. Egido, and S. Mezzano. 2014. Gremlin activates the Smad pathway linked to epithelial mesenchymal transdifferentiation in cultured tubular epithelial cells. BioMed research international 2014
Sethi, A., A. Jain, G. S. Zode, R. J. Wordinger, and A. F. Clark. 2011. Role of TGFβ/Smad signaling in gremlin induction of human trabecular meshwork extracellular matrix proteins. Investigative ophthalmology & visual science 52(8):5251-5259.
Sherr, C. J., and J. M. Roberts. 1995. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes & development 9(10):1149-1163.
Sherr, C. J., and J. M. Roberts. 1999. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & development 13(12):1501-1512.
Shi, Y., and J. Massagué. 2003. Mechanisms of TGF-β signaling from cell membrane to the nucleus. Cell 113(6):685-700.
Shimasaki, S., R. K. Moore, F. Otsuka, and G. F. Erickson. 2004. The bone morphogenetic protein system in mammalian reproduction. Endocrine reviews 25(1):72-101.
Slingerland, J., and M. Pagano. 2000. Regulation of the cdk inhibitor p27 and its deregulation in cancer. Journal of cellular physiology 183(1):10-17.
Sneddon, J. B., H. H. Zhen, K. Montgomery, M. van de Rijn, A. D. Tward, R. West, H. Gladstone, H. Y. Chang, G. S. Morganroth, and A. E. Oro. 2006. Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation. Proceedings of the national academy of sciences 103(40):14842-14847.
Sun, J., F.-F. Zhuang, J. E. Mullersman, H. Chen, E. J. Robertson, D. Warburton, Y.-H. Liu, and W. Shi. 2006. BMP4 activation and secretion are negatively regulated by an intracellular gremlin-BMP4 interaction. Journal of Biological Chemistry 281(39):29349-29356.
Topol, L. Z., B. Bardot, Q. Zhang, J. Resau, E. Huillard, M. Marx, G. Calothy, and D. G. Blair. 2000. Biosynthesis, post-translation modification, and functional characterization of Drm/Gremlin. Journal of Biological Chemistry 275(12):8785-8793.
Wordinger, R. J., R. Agarwal, M. Talati, J. Fuller, W. Lambert, and A. F. Clark. 2002. Expression of bone morphogenetic proteins (BMP), BMP receptors, and BMP associated proteins in human trabecular meshwork and optic nerve head cells and tissues. Mol Vis 8(2):241-250.
Wordinger, R. J., and A. F. Clark. 2007. Bone morphogenetic proteins and their receptors in the eye. Experimental Biology and Medicine 232(8):979-992.
Wordinger, R. J., D. L. Fleenor, P. E. Hellberg, I.-H. Pang, T. O. Tovar, G. S. Zode, J. A. Fuller, and A. F. Clark. 2007. Effects of TGF-β2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma. Investigative ophthalmology & visual science 48(3):1191-1200.
Wordinger, R. J., G. Zode, and A. F. Clark. 2008. Focus on molecules: gremlin. Experimental eye research 87(2):78.
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