進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-0709201019334600
論文名稱(中文) 探討Oct4在肺癌中調控Egr-1的角色
論文名稱(英文) Roles of Oct4 in Regulation of Egr-1 in Lung Cancer
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 98
學期 2
出版年 99
研究生(中文) 林碩甫
研究生(英文) Shuo-Fu Lin
學號 s4697411
學位類別 碩士
語文別 中文
論文頁數 57頁
口試委員 指導教授-蕭璦莉
口試委員-吳昭良
口試委員-張孟雅
口試委員-張芝瑞
口試委員-李哲欣
中文關鍵字 肺癌  Egr-1  Oct4  轉移 
英文關鍵字 lung cancer  Egr-1  Oct4  metastasis 
學科別分類
中文摘要 Oct4 被視為保持幹細胞多潛能性的重要分子。在許多的人類腫瘤生成的過程中,Oct-4也扮演了重要的角色。在肺癌細胞中,Oct-4的持續表現使得癌細胞維持幹細胞的特性,使得癌細胞更不容易被殺死。Egr-1 (early growth response gene-1)是一種鋅指結構(zinc-finger)的轉錄分子,已知能夠調控許多基因的表現,包括發炎反應、細胞分化、生長及發育。雖然Egr-1在從前被認為是一種抑癌基因,但是近年來的研究發現,Egr-1其實也具有致癌基因的特性,所以可以說是一種具有雙面功能的基因。Egr-1已知參與上皮-間葉細胞轉換(epithelial-mesenchymal transition,EMT)與血管新生(angiogenesis),並且會活化有關腫瘤轉移的基因,例如osteopontin (OPN)。根據微陣列分析結果,將老鼠胚胎幹細胞的Oct4抑制後,會讓Egr-1 mRNA的表現量下降,在我們的實驗結果也發現轉移程度越高的肺癌細胞株,Egr-1的表現量也越高。Oct4與Egr-1可能在肺癌細胞致病機制與轉移上扮演重要角色,但是二者之間的關係與重要性仍然未知。因此本研究利用轉染方式將Oct4 cDNA送入H1299肺癌細胞,並利用RT-PCR與西方點墨法分析結果,發現Egr-1 RNA與蛋白質隨著Oct4 cDNA的表現量升高而增加。同樣的分析以反轉錄病毒感染而持續表現Oct4的A549細胞株,也可以發現Egr-1 基因的持續表現。接著利用螢光報導基因分析發現,在A549以及H1299肺癌細胞中,Oct4能夠活化Egr-1的啟動子活性,並利用將啟動子分段切除方式與染色質免疫沉澱分析發現Egr-1啟動子上有Oct4的結合位置。利用Boyden chamber分析在Oct4過量表現的A549細胞與H1299細胞,發現在抑制Egr-1的表現後,細胞移動的能力明顯下降。另外,OPN的表現量也在抑制Egr-1的表現後有明顯的下降。在動物實驗中,以靜脈注射的方式打入NOD/SCID小鼠不同處理的A549細胞,觀察癌細胞轉移到肺的情況,結果發現過量表現Oct4並抑制Egr-1的A549細胞,轉移的現象較少。由以上的結果顯示,Oct4參與肺癌發展的過程中,會藉由增加Egr-1的表現而影響其轉移的機制。
英文摘要 Oct4 is regarded as one of the key regulators of stem cell pluripotency. It has been detected in several human cancers, suggesting a potentially critical role in tumorigenesis. Oct4 expression also maintains cancer stem-like properties in lung cancer. Early growth response gene-1 (Egr-1), a zinc-finger transcription factor, has been implicated in the regulation of many genes involved in inflammation, differentiation, growth, and development. Egr-1 is generally thought to play a tumor suppressor function. However, recent studies have shown that Egr-1 is a Janus-faced protein in cancer because it may be involved in epithelial-mesenchymal transition (EMT) and angiogenesis. Egr-1 also upregulates the expression of osteopontin (OPN) that is involved in modulating cell functions and increasing lung cancer cell migration. Previous microarray data revealed that knockdown of Oct4 in embryonic stem cell decreases Egr-1 mRNA expression. Our study also showed that protein levels of Egr-1 were increased in metastatic lung cancer cell lines. Oct4 and Egr-1 may play important roles in the pathogenesis of lung cancer. However, little is known about their relationship and significance in metastatic lung cancer. Therefore, I transfected H1299 human lung cancer cell line H1299 with Oct4 cDNA and examined the expression of Egr-1 by RT-PCR and immunoblot analysis. Expression of Egr-1 was increased in cells stably transduced with Oct4. Same results were also obtained from A549 cells stably overexpressing Oct4 via retrovirus-mediated gene transfer. In addition, luciferase reporter and chromatin immunoprecipitation assays also revealed that Oct4 upregulated Egr-1 expression through direct binding to its promoter. Knockdown of Egr-1 decreased the invasive potential of Oct4-overexpressing A549 and H1299 cells by Boyden chamber analysis. Expression of OPN was also decreased after knockdown of Egr-1 in Oct4-overexpressing A549 cells. Animal studies also showed that knockdown of Egr-1 reduced tumor nodules in the lung after intravenous injection of Oct4-overexpressing A549 cells to NOD/SCID mice. In conclusion, these results suggest that Oct4 may be involved in lung cancer progression and metastasis through upregulation of Egr-1 expression.
論文目次 中文摘要..........II
Abstract.........IV
誌謝........VI
目錄..........VIII
圖目錄..........X
縮寫..........XII
第一章 緒論..........1
A、肺癌(Lung cancer)..........1
B、癌轉移(tumor metastasis)..........2
C、Oct4..........3
1.概論..........3
2.Oct4與腫瘤..........3
3.Oct4 與癌轉移..........4
4. Oct4 的下游基因..........4
5. Oct4的調控..........5
D、Egr-1..........6
1.概論..........6
2. Egr-1與腫瘤..........6
3.Egr-1與癌轉移..........7
4. Egr-1的調控..........8
5. Oct4與Egr-1..........8
6.Egr-1與Osteopontin(OPN)..........9
E、Osteopontin(OPN)..........10
1.概論..........10
2. OPN與癌轉移..........10
第二章 研究目的..........12
第三章 材料與方法..........13
材料..........13
1.質體..........13
2.細胞株..........13
3.慢病毒..........14
4.實驗動物..........15
5.寡去氧核苷酸(Oligodeoxynucleotides,ODN)..........15
方法..........15
1. 細胞株與細胞培養..........15
2. 慢病毒的生產與感染細胞..........16
3. Stable clone的選殖..........17
4. 西方點墨法分析..........18
5. 反轉錄酶-聚合酶連鎖反應 (Reverse transcriptase-polymerase chain reaction, RT-PCR)..........20
6. 啟動子活性分析..........21
7. Egr-1 啟動子片段切除的建構..........21
8. 細胞移動分析(migration assay)..........22
9. 動物實驗..........23
10. 免疫組織染色(Immunohistochemistry)..........23
11. Enzyme-linked immunosorbent assay(ELISA)..........24
12. 統計學..........25
第四章 結果..........26
一、在肺癌細胞中過量表現Oct4會增加Egr-1蛋白質以及mRNA的表現..........26
二、在肺癌細胞中過量表現Oct4能夠活化Egr-1的啟動子,且發現Egr-1 啟動子上有Oct4的結合位置..........27
三、在肺癌細胞中利用shRNA 抑制Egr-1基因的表現,發現Oct4 mRNA表現量不受影響..........28
四、在動物模式中證明肺癌細胞Oct4過量表現之下Egr-1也會在相同位置的表現..........28
五、Egr-1蛋白質的表現量在轉移程度愈強的肺癌細胞中表現量愈高..........29
六、Egr-1基因剔除的小鼠胚胎纖維母細胞(mouse embryonic fibroblast, MEF) 細胞移動能力明顯低於野生型小鼠MEF..........29
七、過量表現Oct4的肺癌細胞,細胞移動能力會上升,若進一步抑制Egr-1則會降低細胞的移動能力。並在動物模式中證明過量表現Oct4的肺癌細胞會經由Egr-1促進細胞轉移的能力..........30
八、在肺癌細胞中利用shRNA抑制Oct4的表現,Egr-1與Osteopontin (OPN)的mRNA與蛋白質表現也會被抑制。在過量表現Oct4的肺癌細胞A549中進一步抑制Egr-1的基因表現,則會抑制OPN的蛋白質表現。..........31
第五章 總結..........33
第六章 討論..........34
參考文獻..........40
圖..........45
自述..........57
參考文獻 Adamson, E.D., and Mercola, D. (2002). Egr1 transcription factor: multiple roles in prostate tumor cell growth and survival. Tumour Biol 23, 93-102.
Atlasi, Y., Mowla, S.J., Ziaee, S.A., and Bahrami, A.R. (2007). OCT-4, an embryonic stem cell marker, is highly expressed in bladder cancer. Int J Cancer 120, 1598-1602.
Baron, V., Adamson, E.D., Calogero, A., Ragona, G., and Mercola, D. (2006). The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin. Cancer Gene Ther 13, 115-124.
Ben-Shushan, E., Thompson, J.R., Gudas, L.J., and Bergman, Y. (1998). Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site. Mol Cell Biol 18, 1866-1878.
Botquin, V., Hess, H., Fuhrmann, G., Anastassiadis, C., Gross, M.K., Vriend, G., and Scholer, H.R. (1998). New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2. Genes Dev 12, 2073-2090.
Catena, R., Tiveron, C., Ronchi, A., Porta, S., Ferri, A., Tatangelo, L., Cavallaro, M., Favaro, R., Ottolenghi, S., Reinbold, R., et al. (2004). Conserved POU binding DNA sites in the Sox2 upstream enhancer regulate gene expression in embryonic and neural stem cells. J Biol Chem 279, 41846-41857.
Chaivorapol, C., Melton, C., Wei, G., Yeh, R.F., Ramalho-Santos, M., Blelloch, R., and Li, H. (2008). CompMoby: comparative MobyDick for detection of cis-regulatory motifs. BMC Bioinformatics 9, 455.
Chambers, A.F., Wilson, S.M., Kerkvliet, N., O'Malley, F.P., Harris, J.F., and Casson, A.G. (1996). Osteopontin expression in lung cancer. Lung Cancer 15, 311-323.
Chang, C.C., Shieh, G.S., Wu, P., Lin, C.C., Shiau, A.L., and Wu, C.L. (2008). Oct-3/4 expression reflects tumor progression and regulates motility of bladder cancer cells. Cancer Res 68, 6281-6291.
Chellaiah, M., and Hruska, K. (1996). Osteopontin stimulates gelsolin-associated phosphoinositide levels and phosphatidylinositol triphosphate-hydroxyl kinase. Mol Biol Cell 7, 743-753.
Chen, Y.C., Hsu, H.S., Chen, Y.W., Tsai, T.H., How, C.K., Wang, C.Y., Hung, S.C., Chang, Y.L., Tsai, M.L., Lee, Y.Y., et al. (2008). Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS One 3, e2637.
Chen, Z., Xu, W.R., Qian, H., Zhu, W., Bu, X.F., Wang, S., Yan, Y.M., Mao, F., Gu, H.B., Cao, H.L., et al. (2009). Oct4, a novel marker for human gastric cancer. J Surg Oncol 99, 414-419.
Cheng, T., Wang, Y., and Dai, W. (1994). Transcription factor egr-1 is involved in phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells. J Biol Chem 269, 30848-30853.
Chin, D., Boyle, G.M., Kane, A.J., Theile, D.R., Hayward, N.K., Parson, P.G., and Coman, W.B. (2005). Invasion and metastasis markers in cancers. Br J Plast Surg 58, 466-474.
Chu, Y.W., Yang, P.C., Yang, S.C., Shyu, Y.C., Hendrix, M.J., Wu, R., and Wu, C.W. (1997). Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 17, 353-360.
Covello, K.L., Kehler, J., Yu, H., Gordan, J.D., Arsham, A.M., Hu, C.J., Labosky, P.A., Simon, M.C., and Keith, B. (2006). HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev 20, 557-570.
Dann, C.T., Alvarado, A.L., Molyneux, L.A., Denard, B.S., Garbers, D.L., and Porteus, M.H. (2008). Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells 26, 2928-2937.
Ferraro, B., Bepler, G., Sharma, S., Cantor, A., and Haura, E.B. (2005). EGR1 predicts PTEN and survival in patients with non-small-cell lung cancer. J Clin Oncol 23, 1921-1926.
Geiger, T.R., and Peeper, D.S. (2009). Metastasis mechanisms. Biochim Biophys Acta 1796, 293-308.
Grotegut, S., von Schweinitz, D., Christofori, G., and Lehembre, F. (2006). Hepatocyte growth factor induces cell scattering through MAPK/Egr-1-mediated upregulation of Snail. EMBO J 25, 3534-3545.
Horie, M., Sakamoto, K.M., and Broxmeyer, H.E. (1996). Regulation of egr-1 gene expression by retinoic acid in a human growth factor-dependent cell line. Int J Hematol 63, 303-309.
Hu, T., Liu, S., Breiter, D.R., Wang, F., Tang, Y., and Sun, S. (2008). Octamer 4 small interfering RNA results in cancer stem cell-like cell apoptosis. Cancer Res 68, 6533-6540.
Jemal, A., Siegel, R., Xu, J., and Ward, E. (2010). Cancer Statistics, 2010. CA Cancer J Clin.
Karoubi, G., Gugger, M., Schmid, R., and Dutly, A. (2009). OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention. Interact Cardiovasc Thorac Surg 8, 393-397.
Kraft, H.J., Mosselman, S., Smits, H.A., Hohenstein, P., Piek, E., Chen, Q., Artzt, K., and van Zoelen, E.J. (1996). Oct-4 regulates alternative platelet-derived growth factor alpha receptor gene promoter in human embryonal carcinoma cells. J Biol Chem 271, 12873-12878.
Kratz, J.R., Yagui-Beltran, A., and Jablons, D.M. (2010). Cancer stem cells in lung tumorigenesis. Ann Thorac Surg 89, S2090-2095.
Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S.Y., Suemori, H., Nakatsuji, N., and Tada, T. (2005). Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol Cell Biol 25, 2475-2485.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Levina, V., Marrangoni, A.M., DeMarco, R., Gorelik, E., and Lokshin, A.E. (2008). Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties. PLoS One 3, e3077.
Liu, C., Rangnekar, V.M., Adamson, E., and Mercola, D. (1998). Suppression of growth and transformation and induction of apoptosis by EGR-1. Cancer Gene Ther 5, 3-28.
Liu, Q.F., Yu, H.W., and Liu, G.N. (2009). Egr-1 upregulates OPN through direct binding to its promoter and OPN upregulates Egr-1 via the ERK pathway. Mol Cell Biochem 332, 77-84.
Loh, Y.H., Wu, Q., Chew, J.L., Vega, V.B., Zhang, W., Chen, X., Bourque, G., George, J., Leong, B., Liu, J., et al. (2006). The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38, 431-440.
Ma, J., Ren, Z., Ma, Y., Xu, L., Zhao, Y., Zheng, C., Fang, Y., Xue, T., Sun, B., and Xiao, W. (2009). Targeted knockdown of EGR-1 inhibits IL-8 production and IL-8-mediated invasion of prostate cancer cells through suppressing EGR-1/NF-kappaB synergy. J Biol Chem 284, 34600-34606.
Min, I.M., Pietramaggiori, G., Kim, F.S., Passegue, E., Stevenson, K.E., and Wagers, A.J. (2008). The transcription factor EGR1 controls both the proliferation and localization of hematopoietic stem cells. Cell Stem Cell 2, 380-391.
Niwa, H., Miyazaki, J., and Smith, A.G. (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24, 372-376.
Okamoto, K., Okazawa, H., Okuda, A., Sakai, M., Muramatsu, M., and Hamada, H. (1990). A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60, 461-472.
Okuda, A., Fukushima, A., Nishimoto, M., Orimo, A., Yamagishi, T., Nabeshima, Y., Kuro-o, M., Boon, K., Keaveney, M., Stunnenberg, H.G., et al. (1998). UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells. EMBO J 17, 2019-2032.
Parra, E., Ortega, A., and Saenz, L. (2009). Down-regulation of Egr-1 by siRNA inhibits growth of human prostate carcinoma cell line PC-3. Oncol Rep 22, 1513-1518.
Patarca, R., Saavedra, R.A., and Cantor, H. (1993). Molecular and cellular basis of genetic resistance to bacterial infection: the role of the early T-lymphocyte activation-1/osteopontin gene. Crit Rev Immunol 13, 225-246.
Philip, S., Bulbule, A., and Kundu, G.C. (2001). Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-kappa B-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells. J Biol Chem 276, 44926-44935.
Rodda, D.J., Chew, J.L., Lim, L.H., Loh, Y.H., Wang, B., Ng, H.H., and Robson, P. (2005). Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 280, 24731-24737.
Rosner, M.H., Vigano, M.A., Ozato, K., Timmons, P.M., Poirier, F., Rigby, P.W., and Staudt, L.M. (1990). A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345, 686-692.
Saigusa, S., Tanaka, K., Toiyama, Y., Yokoe, T., Okugawa, Y., Ioue, Y., Miki, C., and Kusunoki, M. (2009). Correlation of CD133, OCT4, and SOX2 in rectal cancer and their association with distant recurrence after chemoradiotherapy. Ann Surg Oncol 16, 3488-3498.
Sauer, L., Gitenay, D., Vo, C., and Baron, V.T. (2010). Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells. Oncogene 29, 2628-2637.
Schoorlemmer, J., van Puijenbroek, A., van Den Eijnden, M., Jonk, L., Pals, C., and Kruijer, W. (1994). Characterization of a negative retinoic acid response element in the murine Oct4 promoter. Mol Cell Biol 14, 1122-1136.
Senger, D.R., Perruzzi, C.A., Gracey, C.F., Papadopoulos, A., and Tenen, D.G. (1988). Secreted phosphoproteins associated with neoplastic transformation: close homology with plasma proteins cleaved during blood coagulation. Cancer Res 48, 5770-5774.
Shimoyamada, H., Yazawa, T., Sato, H., Okudela, K., Ishii, J., Sakaeda, M., Kashiwagi, K., Suzuki, T., Mitsui, H., Woo, T., et al. (2010). Early growth response-1 induces and enhances vascular endothelial growth factor-a expression in lung cancer cells. Am J Pathol 177, 70-83.
Shin, S.Y., Kim, J.H., Baker, A., Lim, Y., and Lee, Y.H. (2010). Transcription factor Egr-1 is essential for maximal matrix metalloproteinase-9 transcription by tumor necrosis factor alpha. Mol Cancer Res 8, 507-519.
Sodek, J., Batista Da Silva, A.P., and Zohar, R. (2006). Osteopontin and mucosal protection. J Dent Res 85, 404-415.
Sung, M.T., Jones, T.D., Beck, S.D., Foster, R.S., and Cheng, L. (2006). OCT4 is superior to CD30 in the diagnosis of metastatic embryonal carcinomas after chemotherapy. Hum Pathol 37, 662-667.
Tokuzawa, Y., Kaiho, E., Maruyama, M., Takahashi, K., Mitsui, K., Maeda, M., Niwa, H., and Yamanaka, S. (2003). Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development. Mol Cell Biol 23, 2699-2708.
Tuck, A.B., Hota, C., Wilson, S.M., and Chambers, A.F. (2003). Osteopontin-induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways. Oncogene 22, 1198-1205.
Tureyen, K., Brooks, N., Bowen, K., Svaren, J., and Vemuganti, R. (2008). Transcription factor early growth response-1 induction mediates inflammatory gene expression and brain damage following transient focal ischemia. J Neurochem 105, 1313-1324.
Wai, P.Y., and Kuo, P.C. (2008). Osteopontin: regulation in tumor metastasis. Cancer Metastasis Rev 27, 103-118.
Yamini, B., Yu, X., Gillespie, G.Y., Kufe, D.W., and Weichselbaum, R.R. (2004). Transcriptional targeting of adenovirally delivered tumor necrosis factor alpha by temozolomide in experimental glioblastoma. Cancer Res 64, 6381-6384.
Yu, H.W., Liu, Q.F., and Liu, G.N. (2010). Positive regulation of the Egr-1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGF-beta, ERK, JNK, and p38 MAPK signaling. Biochem Biophys Res Commun 396, 451-456.
Yu, J., Baron, V., Mercola, D., Mustelin, T., and Adamson, E.D. (2007). A network of p73, p53 and Egr1 is required for efficient apoptosis in tumor cells. Cell Death Differ 14, 436-446.
Yuan, H., Corbi, N., Basilico, C., and Dailey, L. (1995). Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Dev 9, 2635-2645.
Zhang, L., Rayner, S., Katoku-Kikyo, N., Romanova, L., and Kikyo, N. (2007). Successful co-immunoprecipitation of Oct4 and Nanog using cross-linking. Biochem Biophys Res Commun 361, 611-614.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2020-12-31起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw