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研究生:游凱棟
研究生(外文):Kai-Dong You
論文名稱:透明細胞型腎細胞癌相關基因:第一型血紅素氧化酶之功能性研究
論文名稱(外文):Studies on the Function of Clear Cell Renal Cell Carcinoma- Associated Gene, Heme Oxygenase-1
指導教授:洪志宏洪志宏引用關係
指導教授(外文):Chih-Hung Hung
學位類別:碩士
校院名稱:元培科學技術學院
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
中文關鍵詞:第一型血紅素氧化酶腎細胞癌
外文關鍵詞:Heme Oxygenase-1Renal Cell Carcinoma
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在2006年,腎細胞癌在佔所有的惡性腫瘤中的百分之三,也是最致命的一種腎臟癌類型。成人腎癌中有超過百分之八十五被診斷為腎細胞癌,其中透明細胞型腎細胞癌為最主要的類型,它對於放射性治療、免疫療法和化療均有高度抗性,所以目前以手術切除為主要的治療方式。因此,我們希望藉由研究腎細胞癌相關基因進而了解其形成的分子機制。在先前的研究中,本實驗室的唐賽文先生建構了腎細胞癌以及正常腎臟組織的全長cDNA資料庫。我們採用即時多重聚合酶鏈反應分析法去分析cDNA資料庫,並比對癌細胞與正常組織之間的差異,發現第一型血紅素氧化酶 (heme oxygenase-1,HO-1) 與血管內皮生長因子A (vascular endothelial growth factor A,VEGF-A)在癌細胞中表現量大於正常組織。
HO-1是一種熱休克蛋白,它能因不同的壓力因子而被誘發,例如重金屬、紫外線、氧化壓力及發炎。HO-1是一種速率限制酵素 (rate-limiting enzyme),它能分解血基質 (heme),並產生一氧化碳 (CO)、亞鐵離子 (Fe2+) 和膽紅素(bilirubin)。 在HEK293細胞株中過度表現HO-1可使VEGF-A的RNA和蛋白質表現量上升。在傷口癒合試驗 (wound healing assay) 與細胞穿透試驗 (transwell assay) 的實驗中發現,穩定表現HO-1的細胞株能促進細胞的遷移。在使用HUVEC細胞株的細胞穿透試驗中,發現穩定表現HO-1的細胞株分泌VEGF-A到培養基中,進而促使HUVEC細胞的遷移。
在訊息傳遞路徑中,我們發現當HO-1表現上升時會增加AKT和ERK1/2的磷酸化。在細胞核中,HO-1參與了NFκB的活化。在細胞存活率試驗中,HO-1可保護細胞抵抗adriamycin。由以上這些結果顯示HO-1表現可增加VEGF-A的分泌,這與腎細胞癌cDNA資料庫的結果類似。總而言之,HO-1在腎細胞癌的發展中可能扮演著癌化的角色,並促進VEGF-A的分泌而造成血管新生。
Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies and is the most lethal of the common urologic cancers in 2006. In adults, RCC accounts for over 85% of all diagnosed renal cancers. The clear cell RCC (ccRCC) is major type in renal cancer. It is high resistance to radiotherapy, immunotherapy and chemotherapy. Surgery remains the most effective method of treating renal cancer. Therefore, to investigate whether tumor associated genes are very important for the molecular mechanisms of RCC, full-length cDNA library of ccRCC and normal kidney constructed by Mr. Sai-Wen Tang was investigated. In the cDNA library, Heme oxygenase 1 (HO-1) and vascular endothelial growth factor A (VEGF-A) were found to be up-reglulated in ccRCC tissues compared to their adjacent normal tissues by real-time polymerase chain reaction.
HO-1 is a heat shock protein and can be induced by various stresses, such as heavy metals, ultraviolet radiation, oxidative stress and inflammation. HO-1 is the rate-limiting enzyme in the degradation of heme into carbon monoxide (CO), iron (Fe2+) and biliverdin. Overexpression of HO-1 is upregulated VEGF-A RNA and protein level in HEK293 cell line. Stable expression of HO-1 is promoted cells migration by wound healing and transwell assay. Using HUVEC tranwell assay it was confirmed that the HO-1 stable clone secreted VEGF-A in the medium to promote HUVEC cells migration. In signal transduction pathways, we found that expression of HO-1 increased the phosphorylation of AKT and ERK 1/2. In nucleus fractionation, HO-1 activated the NFκB expression. By cell viability assay, HO-1 can protect cells which were treated with adriamycin. These results showed that HO-1 expressed was upregulated VEGF-A secretion. Taken together, HO-1 could play a role in the progression of RCC carcinogenesis and promote angiogenesis via VEGF-A secretion.
Table of contents:
摘要 1
Abstract 3
Introduction 5
Materials and Methods 9
1. Plasmid Transformation 9
1.1 Preparation of competent cells 9
1.2 Transformation 9
1.3 Plasmid mini-preparation 10
2. Cell culture 10
3. Transient transfection 11
4. Generating HO-1 stable clones 11
5. RNA extraction and reverse transcription 12
5.1 RNA extraction 12
5.2 Treatment of DNA 12
5.3 Reverse transcription of RNA 13
6. Real-time PCR 14
7. Western Blotting 15
7.1 Preparation of Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 15
7.2 Preparation of cell lysates 16
7.3 Electrophoresis 17
7.4 Semi-dry blotting 18
7.5 Immunoblotting 19
8. Cell viability assay 19
9. Confocal Microscopy 20
9.1 Cells preparation and fixation 20
9.2 Immunostaining 20
9.3 Confocal microscopy 20
10. Wound-healing assay 21
11. Cell Migration Assay for HEK293 cells 21
12. Cell Migration Assay for HUVEC cells 21
13. Heme oxygenase-1 (HO-1) induction by hemin 22
14. Heme oxygenase-1 (HO-1) inhibition by Tin-protoporphyrin IX (SnPP) ……………………………………………………………………………..23
15. Subcelluar fractionation 23
Results 24
Overexpression of HO-1 upregulates VEGF-A mRNA expression in HEK293 cells. 24
VEGF-A protein is upregulated by HO-1 expression in HEK293 cells 24
Hemin treatment increased the VEGF-A protein expression 25
Subcellular localization of stable expression HO-1 protein 26
Stable expression of HO-1 enhanced cell migration 26
Stable expression of HO-1 enhanced HUVEC cells migration 27
An inhibitor of HO-1, Tin-protoporphyrin IX (SnPP), decreased the VEGF-A expression 28
Increase of phosphorylation of the AKT and ERK1/2 in HO-1 stable cell line ……………………………………………………………………………..28
Increase of NFκB p50 and NFκB p65 translocation into nucleus in HO-1 stable cell line 29
Protection of cells from adriamycin-induced apoptosis by expression of HO-1 29
Discussion 31
Figures 35
References 61
1.Lee, T. Y., Lawen, J., and Gupta, R. "Renal angiosarcoma: a case report and literature review," Can J Urol 14 (2007): 3471-6.
2.Jemal, A., Siegel, R., Ward, E., Murray, T., Xu, J., Smigal, C., and Thun, M. J. "Cancer statistics, 2006," CA Cancer J Clin 56 (2006): 106-30.
3.Junker, K., Hindermann, W., von Eggeling, F., Diegmann, J., Haessler, K., and Schubert, J. "CD70: a new tumor specific biomarker for renal cell carcinoma," J Urol 173 (2005): 2150-3.
4.Bruno, J. J., 2nd, Snyder, M. E., Motzer, R. J., and Russo, P. "Renal cell carcinoma local recurrences: impact of surgical treatment and concomitant metastasis on survival," BJU Int 97 (2006): 933-8.
5.Tang, S. W., Chang, W. H., Chao, Y. W., Lin, C. Y., Chen, H. F., Lai, Y. H., Zhan, B. W., Su, Y. C., Jane, S. W., Chen, Y. C., Hsu, C. I., Lin, W. C., Wang, K. C., Lai, M. K., and Lin, J. Y. "Identification of differentially expressed genes in clear cell renal cell carcinoma by analysis of full-length enriched cDNA library," J Biomed Sci 13 (2006): 233-40.
6.Ryter, S. W., Alam, J., and Choi, A. M. "Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications," Physiol Rev 86 (2006): 583-650.
7.Tenhunen, R., Marver, H. S., and Schmid, R. "Microsomal heme oxygenase. Characterization of the enzyme," J Biol Chem 244 (1969): 6388-94.
8.Morse, D., and Choi, A. M. "Heme oxygenase-1: from bench to bedside," Am J Respir Crit Care Med 172 (2005): 660-70.
9.Durante, W., Christodoulides, N., Cheng, K., Peyton, K. J., Sunahara, R. K., and Schafer, A. I. "cAMP induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle," Am J Physiol 273 (1997): H317-23.
10.Lash, G. E., McLaughlin, B. E., MacDonald-Goodfellow, S. K., Smith, G. N., Brien, J. F., Marks, G. S., Nakatsu, K., and Graham, C. H. "Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta," Am J Physiol Heart Circ Physiol 284 (2003): H160-7.
11.Liu, X. M., Peyton, K. J., Ensenat, D., Wang, H., Schafer, A. I., Alam, J., and Durante, W. "Endoplasmic reticulum stress stimulates heme oxygenase-1 gene expression in vascular smooth muscle. Role in cell survival," J Biol Chem 280 (2005): 872-7.
12.Favatier, F., and Polla, B. S. "Tobacco-smoke-inducible human haem oxygenase-1 gene expression: role of distinct transcription factors and reactive oxygen intermediates," Biochem J 353 (2001): 475-82.
13.Schnickel, G. T., Hsieh, G. R., Kachikwu, E. L., Garcia, C., Shefizadeh, A., Fishbein, M. C., and Ardehali, A. "Cytoprotective gene HO-1 and chronic rejection in heart transplantation," Transplant Proc 38 (2006): 3259-62.
14.Waller, H. L., Harper, S. J., Hosgood, S. A., Bagul, A., Kay, M. D., Kaushik, M., Yang, B., Bicknell, G. R., and Nicholson, M. L. "Differential expression of cytoprotective and apoptotic genes in an ischaemia-reperfusion isolated organ perfusion model of the transplanted kidney," Transpl Int (2007).
15.Kim, J. H., Yang, J. I., Jung, M. H., Hwa, J. S., Kang, K. R., Park, D. J., Roh, G. S., Cho, G. J., Choi, W. S., and Chang, S. H. "Heme oxygenase-1 protects rat kidney from ureteral obstruction via an antiapoptotic pathway," J Am Soc Nephrol 17 (2006): 1373-81.
16.Kim, H. P., Wang, X., Zhang, J., Suh, G. Y., Benjamin, I. J., Ryter, S. W., and Choi, A. M. "Heat shock protein-70 mediates the cytoprotective effect of carbon monoxide: involvement of p38 beta MAPK and heat shock factor-1," J Immunol 175 (2005): 2622-9.
17.Neuzil, J., and Stocker, R. "Bilirubin attenuates radical-mediated damage to serum albumin," FEBS Lett 331 (1993): 281-4.
18.Neuzil, J., and Stocker, R. "Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation," J Biol Chem 269 (1994): 16712-9.
19.Liu, Z. M., Chen, G. G., Ng, E. K., Leung, W. K., Sung, J. J., and Chung, S. C. "Upregulation of heme oxygenase-1 and p21 confers resistance to apoptosis in human gastric cancer cells," Oncogene 23 (2004): 503-13.
20.Yanagawa, T., Omura, K., Harada, H., Nakaso, K., Iwasa, S., Koyama, Y., Onizawa, K., Yusa, H., and Yoshida, H. "Heme oxygenase-1 expression predicts cervical lymph node metastasis of tongue squamous cell carcinomas," Oral Oncol 40 (2004): 21-7.
21.Mayerhofer, M., Florian, S., Krauth, M. T., Aichberger, K. J., Bilban, M., Marculescu, R., Printz, D., Fritsch, G., Wagner, O., Selzer, E., Sperr, W. R., Valent, P., and Sillaber, C. "Identification of heme oxygenase-1 as a novel BCR/ABL-dependent survival factor in chronic myeloid leukemia," Cancer Res 64 (2004): 3148-54.
22.Flad, T., Mueller, L., Dihazi, H., Grigorova, V., Bogumil, R., Beck, A., Thedieck, C., Mueller, G. A., Kalbacher, H., and Mueller, C. A. "T cell epitope definition by differential mass spectrometry: identification of a novel, immunogenic HLA-B8 ligand directly from renal cancer tissue," Proteomics 6 (2006): 364-74.
23.Berberat, P. O., Dambrauskas, Z., Gulbinas, A., Giese, T., Giese, N., Kunzli, B., Autschbach, F., Meuer, S., Buchler, M. W., and Friess, H. "Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment," Clin Cancer Res 11 (2005): 3790-8.
24.Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., Fiddes, J. C., and Abraham, J. A. "The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing," J Biol Chem 266 (1991): 11947-54.
25.Ferrara, N. "Vascular endothelial growth factor: basic science and clinical progress," Endocr Rev 25 (2004): 581-611.
26.Risau, W. "Mechanisms of angiogenesis," Nature 386 (1997): 671-4.
27.Schneider, B. P., and Miller, K. D. "Angiogenesis of breast cancer," J Clin Oncol 23 (2005): 1782-90.
28.Herbst, R. S., Onn, A., and Sandler, A. "Angiogenesis and lung cancer: prognostic and therapeutic implications," J Clin Oncol 23 (2005): 3243-56.
29.Kabbinavar, F., Hurwitz, H. I., Fehrenbacher, L., Meropol, N. J., Novotny, W. F., Lieberman, G., Griffing, S., and Bergsland, E. "Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer," J Clin Oncol 21 (2003): 60-5.
30.Rmali, K. A., Puntis, M. C., and Jiang, W. G. "Tumour-associated angiogenesis in human colorectal cancer," Colorectal Dis 9 (2007): 3-14.
31.Kosharskyy, B., Solban, N., Chang, S. K., Rizvi, I., Chang, Y., and Hasan, T. "A mechanism-based combination therapy reduces local tumor growth and metastasis in an orthotopic model of prostate cancer," Cancer Res 66 (2006): 10953-8.
32.Guerrin, M., Moukadiri, H., Chollet, P., Moro, F., Dutt, K., Malecaze, F., and Plouet, J. "Vasculotropin/vascular endothelial growth factor is an autocrine growth factor for human retinal pigment epithelial cells cultured in vitro," J Cell Physiol 164 (1995): 385-94.
33.Sondell, M., Lundborg, G., and Kanje, M. "Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system," J Neurosci 19 (1999): 5731-40.
34.Oberg-Welsh, C., Sandler, S., Andersson, A., and Welsh, M. "Effects of vascular endothelial growth factor on pancreatic duct cell replication and the insulin production of fetal islet-like cell clusters in vitro," Mol Cell Endocrinol 126 (1997): 125-32.
35.Baldewijns, M. M., Thijssen, V. L., Van den Eynden, G. G., Van Laere, S. J., Bluekens, A. M., Roskams, T., van Poppel, H., De Bruine, A. P., Griffioen, A. W., and Vermeulen, P. B. "High-grade clear cell renal cell carcinoma has a higher angiogenic activity than low-grade renal cell carcinoma based on histomorphological quantification and qRT-PCR mRNA expression profile," Br J Cancer 96 (2007): 1888-95.
36.Favot, L., Keravis, T., Holl, V., Le Bec, A., and Lugnier, C. "VEGF-induced HUVEC migration and proliferation are decreased by PDE2 and PDE4 inhibitors," Thromb Haemost 90 (2003): 334-43.
37.Kim, H. J., So, H. S., Lee, J. H., Lee, J. H., Park, C., Park, S. Y., Kim, Y. H., Youn, M. J., Kim, S. J., Chung, S. Y., Lee, K. M., and Park, R. "Heme oxygenase-1 attenuates the cisplatin-induced apoptosis of auditory cells via down-regulation of reactive oxygen species generation," Free Radic Biol Med 40 (2006): 1810-9.
38.Tewey, K. M., Rowe, T. C., Yang, L., Halligan, B. D., and Liu, L. F. "Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II," Science 226 (1984): 466-8.
39.Jazwa, A., Loboda, A., Golda, S., Cisowski, J., Szelag, M., Zagorska, A., Sroczynska, P., Drukala, J., Jozkowicz, A., and Dulak, J. "Effect of heme and heme oxygenase-1 on vascular endothelial growth factor synthesis and angiogenic potency of human keratinocytes," Free Radic Biol Med 40 (2006): 1250-63.
40.Kim, E. H., Na, H. K., and Surh, Y. J. "Upregulation of VEGF by 15-deoxy-Delta12,14-prostaglandin J2 via heme oxygenase-1 and ERK1/2 signaling in MCF-7 cells," Ann N Y Acad Sci 1090 (2006): 375-84.
41.Fridman, R., Scott, A. F., Muller, D., Reich, R., and Penno, M. B. "The role of cell adhesion and migration in the in vitro invasiveness of mouse adrenal carcinoma cells," Invasion Metastasis 10 (1990): 208-24.
42.Deshane, J., Chen, S., Caballero, S., Grochot-Przeczek, A., Was, H., Li Calzi, S., Lach, R., Hock, T. D., Chen, B., Hill-Kapturczak, N., Siegal, G. P., Dulak, J., Jozkowicz, A., Grant, M. B., and Agarwal, A. "Stromal cell-derived factor 1 promotes angiogenesis via a heme oxygenase 1-dependent mechanism," J Exp Med 204 (2007): 605-18.
43.Dulak, J., Jozkowicz, A., Foresti, R., Kasza, A., Frick, M., Huk, I., Green, C. J., Pachinger, O., Weidinger, F., and Motterlini, R. "Heme oxygenase activity modulates vascular endothelial growth factor synthesis in vascular smooth muscle cells," Antioxid Redox Signal 4 (2002): 229-40.
44.Oh, H. K., Ha, J. M., O, E., Lee, B. H., Lee, S. K., Shim, B. S., Hong, Y. K., and Joe, Y. A. "Tumor angiogenesis promoted by ex vivo differentiated endothelial progenitor cells is effectively inhibited by an angiogenesis inhibitor, TK1-2," Cancer Res 67 (2007): 4851-9.
45.Zhou, G. L., Tucker, D. F., Bae, S. S., Bhatheja, K., Birnbaum, M. J., and Field, J. "Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration," J Biol Chem 281 (2006): 36443-53.
46.Zhu, D., Hattori, H., Jo, H., Jia, Y., Subramanian, K. K., Loison, F., You, J., Le, Y., Honczarenko, M., Silberstein, L., and Luo, H. R. "Deactivation of phosphatidylinositol 3,4,5-trisphosphate/Akt signaling mediates neutrophil spontaneous death," Proc Natl Acad Sci U S A 103 (2006): 14836-41.
47.Perez-Pinera, P., Menendez-Gonzalez, M., del Valle, M., and Vega, J. A. "Sodium chloride regulates Extracellular Regulated Kinase 1/2 in different tumor cell lines," Mol Cell Biochem 293 (2006): 93-101.
48.Rajalingam, K., Wunder, C., Brinkmann, V., Churin, Y., Hekman, M., Sievers, C., Rapp, U. R., and Rudel, T. "Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration," Nat Cell Biol 7 (2005): 837-43.
49.Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D. B. "NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase," Nature 401 (1999): 82-5.
50.Tracz, M. J., Alam, J., and Nath, K. A. "Physiology and pathophysiology of heme: implications for kidney disease," J Am Soc Nephrol 18 (2007): 414-20.
51.Akagi, R., Takahashi, T., and Sassa, S. "Cytoprotective effects of heme oxygenase in acute renal failure," Contrib Nephrol 148 (2005): 70-85.
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