Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20734
標題: 利用核磁共振法進行非小細胞肺癌代謝體學分析之研究
Metabolomics Analysis in Non-Small Cell Lung Cancer by NMR Approach
作者: 楊明錡
Yang, Ming-Chi
關鍵字: 代謝體學;Metabolomics;肺癌;核磁共振;主成分分析;Lung cancer;Nuclear magnetic resonance;Principal component analysis
出版社: 分子生物學研究所
引用: 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA: A Cancer Journal for Clinicians 2012;62:10-29. 2. Terri A, Rick A, Priti B, Durado B, Amy C, Vilma C, et al. Cancer fact & figures 2012. American Cancer Society 2012. 3. Hassanein M, Callison J, Callaway-Lane C, Aldrich M, Grogan E, Massion P. The state of molecular biomarkers for the early detection of lung cancer. Cancer Prevention Research 2012;5:992-1006. 4. Hori S, Nishiumi S, Kobayashi K, Shinohara M, Hatakeyama Y, Kotani Y, et al. A metabolomic approach to lung cancer. Lung cancer 2011;74:284-92. 5. Collins. LG, Christofer H, Robert P, Robert E E. Lung cancer diagnosis and management. American Family Physician 2007;75:56-63. 6. Parkin D. Tobacco-attributable cancer burden in the UK in 2010. British Journal of Cancer 2011;105 Suppl 2:S6-S13. 7. Gorlova O, Weng S, Zhang Y, Amos C, Spitz M. Aggregation of cancer among relatives of never-smoking lung cancer patients. International Journal of Cancer 2007;121:111-8. 8. Bilello K, Murin S, Matthay R. Epidemiology, etiology, and prevention of lung cancer. Clinics in Chest Medicine 2002;23:1-25. 9. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. World Health Organization; 2002. 10. Besaratinia A, Pfeifer G, P. Second-hand smoke and human lung cancer. The Lancet Oncology 2008;9:657-66. 11. Zhu B, Heeschen C, Sievers R, Karliner J, Parmley W, Glantz S, et al. Second hand smoke stimulates tumor angiogenesis and growth. Cancer Cell 2003;4:191-6. 12. Wen C, Lee H. Environmental exposure and lung cancer among nonsmokers: an example of Taiwanese female lung cancer. Journal of Environmental Science and Hhealth Part C, Environmental Carcinogenesis & Ecotoxicology Reviews 2003;21:1-28. 13. Raaschou-Nielsen O, Andersen Z, Hvidberg M, Jensen S, Ketzel M, Sorensen M, et al. Lung cancer incidence and long-term exposure to air pollution from traffic. Environmental Health Perspectives 2011;119:860-5. 14. Turner M, Krewski D, 3rd. PC, Chen Y, Gapstur S, Thun M. Long-term ambient fine particulate matter air pollution and lung cancer in a large cohort of never-smokers. American Journal of Respiratory and Critical Care Medicine 2011;184:1374-81. 15. Hsiung C, Lan Q, Hong Y, Chen C, Hosgood H, Chang I, et al. The 5p15.33 locus is associated with risk of lung adenocarcinoma in never-smoking females in Asia. PLoS Genetics 2010;6. 16. Hosgood H, 3rd., Wang W, Hong Y, Wang J, Chen K, Chang I, et al. Genetic variant in TP63 on locus 3q28 is associated with risk of lung adenocarcinoma among never-smoking females in Asia. Human Genetics 2012;131:1197-203. 17. Lan Q, Hsiung C, Matsuo K, Hong Y, Seow A, Wang Z, et al. Genome-wide association analysis identifies new lung cancer susceptibility loci in never-smoking women in Asia. Nature Genetics 2012;44:1330-5. 18. Hoffman P, Mauer A, Vokes E. Lung cancer. The Lancet 2000;355:479-85. 19. Isaiah JF. The pathogenesis of cancer metastasis the ''seed and soil'' hypothesis revisited. Nature Reviews Cancer 2003;3:1-6. 20. Chaffer C, Weinberg R. A perspective on cancer cell metastasis. Science 2011;331:1559-64. 21. Ritossa P. Problems of prophylactic vaccinations of infants. Rivista dell''Istituto Sieroterapico Italiano 1962;37:79-108. 22. Santoro M. Heat shock factors and the control of the stress response. Biochemical Pharmacology 2000;59:55-63. 23. Rylander M, Feng Y, Bass J, Diller K. Thermally induced injury and heat-shock protein expression in cells and tissues. Annals of the New York Academy of Sciences 2005;1066:222-42. 24. Mitra A, Shevde L, Samant R. Multi-faceted role of HSP40 in cancer. Clinical and Experimental Metastasis 2009;26:559-67. 25. Latchman D. Heat shock proteins and cardiac protection. Cardiovascular Research 2001;51:637-46. 26. Neckers L, Ivy S. Heat shock protein 90. Current Opinion in Oncology 2003;15:419-24. 27. Dragovic Z, Broadley S, Shomura Y, Bracher A, Hartl F. Molecular chaperones of the Hsp110 family act as nucleotide exchange factors of Hsp70s. The EMBO Journal 2006;25:2519-28. 28. Romano C, Benedetto N, Catania M, Rizzo A, Galle F, Losi E, et al. Commonly used antibiotics induce expression of Hsp 27 and Hsp 60 and protect human lymphocytes from apoptosis. International Immunopharmacology 2004;4:1067-73. 29. Kelley W, Georgopoulos C. Chaperones and protein folding. Current opinion in cell biology. 1992;4:984-91. 30. Lindquist S, Craig E. The heat-shock proteins. Annual Review of Genetics 1988;22:631-77. 31. Barnes J, Dix D, Collins B, Luft C, Allen J. Expression of inducible Hsp70 enhances the proliferation of MCF-7 breast cancer cells and protects against the cytotoxic effects of hyperthermia. Cell Stress Chaperones 2001;6:316-25. 32. Garrido C, Schmitt E, Cande C, Vahsen N, Parcellier A, Kroemer G. HSP27 and HSP70: potentially oncogenic apoptosis inhibitors. Cell Cycle 2003;2:579-84. 33. Pick E, Kluger Y, Giltnane J, Moeder C, Camp R, Rimm D, et al. High HSP90 expression is associated with decreased survival in breast cancer. Cancer Research 2007;67:2932-7. 34. Shimamura T, Li D, Ji H, Haringsma H, Liniker E, Borgman C, et al. Hsp90 inhibition suppresses mutant EGFR-T790M signaling and overcomes kinase inhibitor resistance. Cancer Research 2008;68:5827-38. 35. Concannon C, Gorman A, Samali A. On the role of Hsp27 in regulating apoptosis. Apoptosis. An International Journal on Programmed Cell Death 2003;8:61-70. 36. Bausero M, Page D, Osinaga E, Asea A. Surface expression of Hsp25 and Hsp72 differentially regulates tumor growth and metastasis. Tumor Biology 2004;25:243-51. 37. Schwartz B, Shoseyov O, Melnikova V, McCarty M, Leslie M, Roiz L, et al. ACTIBIND, a T2 RNase, competes with angiogenin and inhibits human melanoma growth, angiogenesis, and metastasis. Cancer Research 2007;67:5258-66. 38. Wang F, Feng M, Xu P, Xiao H, Niu P, Yang X, et al. The level of Hsp27 in lymphocytes is negatively associated with a higher risk of lung cancer. Cell Stress Chaperones 2009;14:245-51. 39. Wang X, Li Y, Manjili M, Repasky E, Pardoll D, Subjeck J. Hsp110 over-expression increases the immunogenicity of the murine CT26 colon tumor. Cancer Immunology, Immunotherapy 2002;51:311-9. 40. Sterrenberg J, Blatch G, Edkins A. Human DNAJ in cancer and stem cells. Cancer Letters 2011;312:129-42. 41. Sterrenberg J, latch G, dkins A. Human DNAJ in cancer and stem cells. Cancer Letters 2011;312:129-42. 42. Ohtsuka K. Cloning of a cDNA for heat-shock protein hsp40, a human homologue of bacterial DnaJ. Biochemical and Biophysical Research Communications 1993;197:235~40. 43. Hoe K, Won M, Chung K, Jang Y, Lee S, Kim D, et al. Isolation of a new member of DnaJ-like heat shock protein 40 (Hsp40) from human liver. Biochim Biophys Acta 1998. 44. Chen J, Peck K, Hong T, Yang S, Sher Y, Shih J, et al. Global analysis of gene expression in invasion by a lung cancer model. Cancer Research 2001;61:5223-30. 45. Wang C, Tsai M, Hong T, hang G, Chen C, Yang W, et al. The transcriptional factor YY1 upregulates the novel invasion suppressor HLJ1 expression and inhibits cancer cell invasion. Oncogene 2005;24:4081-93. 46. Tsai M, Wang C, Chang G, Chen C, Chen H, Cheng C, et al. A new tumor suppressor DnaJ-like heat shock protein, HLJ1, and survival of patients with non-small-cell lung carcinoma. Journal of the National Cancer Institute 2006;98:825-38. 47. Wang C, Tsai M, Dai T, Hong T, Chan W, Chen J, et al. Synergistic activation of the tumor suppressor, HLJ1, by the transcription factors YY1 and activator protein 1. Cancer Research 2007;67:4816-26. 48. Chen C, Lin H, Chuang S, Lin S, Chen J. Acidic stress facilitates tyrosine phosphorylation of HLJ1 to associate with actin cytoskeleton in lung cancer cells. Experimental Cell Research 2010;316:2910-21. 49. Lin S, Hsueh C, Yu S, Su C, Shum W, Yeh K, et al. HLJ1 is a novel caspase-3 substrate and its expression enhances UV-induced apoptosis in non-small cell lung carcinoma. Nucleic Acids Research 2010;38:6148-58. 50. Chang T, Yu S, Lin S, Hsiao Y, Chang G, Yang P, et al. Tumor suppressor HLJ1 binds and functionally alters nucleophosmin via activating enhancer binding protein 2alpha complex formation. Cancer Research 2010;70:1656-67. 51. Wishart D. Current progress in computational metabolomics. Briefings in Bioinformatics 2007;8:279-93. 52. Dunn W, Bailey N, Johnson H. Measuring the metabolome: current analytical technologies. The Analyst 2005;130:606-25. 53. Ryan D, Robards K. Metabolomics: The greatest omics of them all? Analytical Chemistry 2006;78:7954-8. 54. Nicholson J, Lindon J, Holmes E. "Metabonomics" : understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 1999;29:1181-9. 55. Griffin J. Metabonomics: NMR spectroscopy and pattern recognition analysis of body fluids and tissues for characterisation of xenobiotic toxicity and disease diagnosis. Current Opinion in Chemical Biology 2003;7:648-54. 56. Clarke C, Haselden J. Metabolic profiling as a tool for understanding mechanisms of toxicity. Toxicologic Pathology 2008;36:140-7. 57. Ioannis P, Anastassios T, Vassiliki E, Klimentini B. Nuclear magneticrResonance (NMR) spectroxcopy: basic principles and phenomena, and their applications to chemistry, biology and medicine. Chemistry Education: Research and Practice in Europe 2002;3:229-52. 58. Nicholson J, Connelly J, Lindon J, Holmes E. Metabonomics: a platform for studying drug toxicity and gene function. Nature Reviews Drug Discovery 2002;153-161. 59. Smolinska A, Blanchet L, Buydens L, Wijmenga S. NMR and pattern recognition methods in metabolomics: from data acquisition to biomarker discovery: a review. Analytica Chimica Acta 2012;750:82-97. 60. Doreen J, Yi, Ng., Kishore K, Pasikanti., Eric C, Yong, Chan. Trend analysis of metabonomics and systematic review of metabonomics-derived cancer marker metabolites. Metabolomics 2010;7:155-78. 61. Gowda G, Zhang S, Gu H, Asiago V, Shanaiah N, Raftery D. Metabolomics-based methods for early disease diagnostics. Expert Review of Molecular Diagnostics 2008;8:617-33. 62. Pearson K. On lines and planes of closest fit to systems of points in space. Philosophical Magazine 1901;2:559-72. 63. Herve A, Williams L. Principal component analysis. Wiley Interdisciplinary Reviews: Computational Statistics 2010;2:433-59. 64. Viant M. Revealing the metabolome of animal tissues using 1H nuclear magnetic resonance spectroscopy. Metabolomics: Methods and Protocols 2007;358:229-46. 65. De Meyer T, Sinnaeve D, Van Gasse B, Rietzschel E, De Buyzere M, Langlois M, et al. Evaluation of standard and advanced preprocessing methods for the univariate analysis of blood serum 1H-NMR spectra. Analytical and Bioanalytical Chemistry 2010;398:1781-90. 66. Liu C, Lin C, Chen W, Chen H, Chang P, Chen J, et al. CRSD: a comprehensive web server for composite regulatory signature discovery. Nucleic Acids Research 2006;34:W571-7. 67. Jackson A, Bartz S, Schelter J, Kobayashi S, Burchard J, Mao M, et al. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnology 2003;21:635-8. 68. Amarzguioui M, Rossi J, Kim D. Approaches for chemically synthesized siRNA and vector-mediated RNAi. FEBS Letters 2005;579:5974-81. 69. Mark R, Viant. Revealing the metabolome of animal tissues using 1H nuclear magnetic resonance spectroscopy. Methods in Molecular Biology 358:229-46. 70. Beltran A, Suarez M, Rodriguez M, Vinaixa M, Samino S, Arola L, et al. Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics. Metabolomics 2007;3:55-67.
摘要: 
HLJ1又被稱為DnaJB4,是屬於熱休克蛋白40家族的成員之一。在先前的研究當中指出HLJ1的表現會與非小細胞肺癌的侵襲、移動以及增生能力呈現負相關,然而目前HLJ1在代謝上的角色則尚未清楚。最近幾年來代謝體學蓬勃發展,是一個能夠深入探討細胞中全代謝物圖譜變化的領域,因此本研究主要是建立一個研究代謝體學的方法並且探討靜默HLJ1後對於癌細胞的代謝圖譜的影響。首先,我們利用病毒感染的方式將HLJ1的shRNA送入肺癌細胞CL1-0中,建立一個靜默HLJ1的穩定細胞株。接著,為了觀察生物體內代謝體的真實狀態,我們從HLJ1基因剔除鼠中取出若干臟器,包括心、肝、肺、腎,來進行代謝體學的分析。西方墨點法顯示,無論在老鼠臟器或是細胞中,HLJ1的表現量明顯的減少。進一步以以1H核磁共振 (NMR)儀分析老鼠臟器及靜默細胞株,並利用主成分分析法對所有樣品進行分群。在此論文中,我們已成功地建立以NMR儀器來分析臟器及細胞中的代謝圖譜,並發現甲醇萃取極性層物質較三氯甲烷所萃取的非極性層,更能將臟器內的代謝物質進行分群。由分析結果顯示,細胞培養液及細胞萃取液經由代謝體學分析後,正常表現HLJ1及靜默HLJ1的組別之間並沒有顯著性差異,然而老鼠的臟器萃取液則存在著些許差異。本研究所得到的代謝體學的實驗結果未來可與靜默HLJ1肺癌細胞的微陣列資料合併分析,相信可以幫助我們更快速的找到與HLJ1相關的目標代謝路徑。

HLJ1, also known as DnaJB4, is a member of DnaJ-like heat shock protein 40 family. The previous studies had shown that HLJ1 expression is negatively correlated with invasion, migration, and proliferation in Non-Small Cell Lung Cancer (NSCLC). However, its role in metabolism is still unknown. Metabolomics has been vigorously developed in recent years, which is a powerful tool to investigate the changes in global metabolite profiles of cells. Therefore this study aims to set up the metabolomics approach and investigate the metabolite profiles of cancer cells with HLJ1 silence. First, the CL1-0 lung cancer cells were transduced with HLJ1 shRNA lentivirus to establish HLJ1-silenced stable cell clones. In addition, to get into the real situation of in vivo metabolomics, several organs from HLJ1 knockout mice, including lung, liver, kidney and heart, were applied to perform metabolomic analysis. Western blotting revealed that HLJ1 expression is significantly silenced no matter mice organs or cultured cell lysates. The metabolite profiles of each sample were detected using 1H nuclear magnetic resonance (NMR) spectrometry and analyzed by principal components analysis (PCA) for metabolite classification. In this study, we have successfully built up NMR-based analysis the metabolite profile of the organ and cell. We found that the classification of the hydrophilic material extracted by methanol is better than that of hydrophobic material by chloroform. Our results also showed that there are few differences in metabolomic profiles between HLJ1 expression and silence groups in mice, but no markedly difference in the cultured media and cell lysate extracts. Furthermore, the metabolomic results may combine with the microarray data of HLJ1-silenced cancer cells in the future, which might help us rapidly identify the targeted metabolic pathways that HLJ1 may be involved in.
URI: http://hdl.handle.net/11455/20734
其他識別: U0005-0702201322310100
Appears in Collections:分子生物學研究所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.