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系統識別號 U0026-0812200915352589
論文名稱(中文) 尋找肝癌的生物標記
論文名稱(英文) Biomarker Discovery in Hepatocellular Carcinoma
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 黃浩一
研究生(英文) Hao-Yi Huang
電子信箱 wwfwcwecwxfl@yaoo.com.tw
學號 T1696112
學位類別 碩士
語文別 英文
論文頁數 82頁
口試委員 指導教授-周楠華
口試委員-劉校生
口試委員-陳淑慧
口試委員-蔡弘文
中文關鍵字 蛋白質體學  介質輔助雷射脫附離子化  生物標記  肝癌 
英文關鍵字 Biomarker  Hepatocellular carcinoma  Proteomics  Matrix-assisted laser desorption/ ionization 
學科別分類
中文摘要 肝癌是人類最具侵略性的癌症之一。大部分的肝癌病人被診斷出來的時候都是在癌症晚期時,因此病人五年內的存活率小於百分之五。為了提高病人的存活機會,早期診斷發現和適當的治療來說就十分重要。蛋白質體學是一種系統性搜尋和疾病相關的蛋白質群之方法。介質輔助雷射脫附離子化 (MALDI)在質譜分析中是屬於軟性雷射脫附法,可用來分析生物標記分子。最近的研究報導指出,利用此一技術可以成功找到癌症的生物標記。為了尋找導致肝癌的新穎生物標記,我們使用MALDI-imaging和SDS-PAGE分析確認的肝癌及週遭之正常組織。我們總共收集了二十位肝癌組織及正常組織配對之肝癌病人的質譜圖。利用這個方法,我們發現在B型肝癌帶原者肝癌組織中,大約在5-6 kDa,11-12 kDa和13-15 kDa會有升高之特殊質譜訊號。此外,在中度分化之肝癌病人肝癌組織質譜圖中,可以發現8-9 kDa和15-16 kDa升高之特殊質譜訊號。從以上這些結果顯示,這些蛋白質分子量之區域可能存在具有潛力的肝癌致癌基因。 我們同時也利用梯度SDS-PAGE,進一步分析同組病人之肝癌和正常組織,並且和MALDI-imaging的結果做對照。最後在Coomassie blue和銀染色的膠體上,針對有興趣分子量的band進行膠體內水解, 配合串聯質譜儀的定序分析。 我們總計發現幾個很有趣的基因,包含ribonuclease UK114,ubiquitin specific proteases 47 (USP47),progesterone receptor membrane component 1 (PGRMC1)和Galectin-4 (Gal-4)。進一步分析肝癌組織後發現,UK114的mRNA在超過一半的肝癌中有被抑制的情況。這結果和最近發表的一篇論文類似: UK114在肝癌組織及肝癌細胞株中表現量會被顯著地抑制。然而USP47的mRNA在一半的肝癌中有比較高的表現。所以USP47在肝癌致癌過程中可能扮演著一個特定的角色。此外,PGRMC1的mRNA在一半的肝癌組織中有被抑制的情形產生。 相反的,PGRMC1的mRNA則在另外一半的肝癌中有比較高的表現。有趣的是PGRMC1 mRNA 在肝癌細胞株的表現趨勢和 vascular endothelial growth factor (VEGF)相當類似。因為VEGF是一個很有名會受到缺氧所誘發之促進血管新生分子,所以目前正在進行一系列實驗,看看PGRMC1是否可以當作缺氧的生物標記。Gal-4的 mRNA 在一半的肝癌組織中有被抑制的現象。然而,其餘的肝癌組織則有相反的表現趨勢。我們未來會釐清Gal-4是否在肝癌之分化及生長扮演重要角色。至於EMP3的部份,我們發現EMP3的 mRNA 表現量在晚期的肝癌細胞株比較高。接著的功能實驗發現,EMP3在HepG2 or HEK293細胞短暫地表現可以抑制細胞的生長。此外,在HEK293細胞內大量表現時,EMP3可以抑制細胞的移動。因此,EMP3對於肝癌來說,可能扮演著抑癌基因的角色。
英文摘要 Hepatocellular carcinoma (HCC) is one of the most common and aggressive human cancers worldwide. Most of HCC patients are diagnosed at advanced stage with 5-year survival rate less than 5%. Therefore, an early diagnosis and appropriate treatment planning for HCC are extremely important. Proteomics is a systematic analysis of protein profile related to human diseases. Matrix-assisted laser desorption/ ionization (MALDI) is a soft ionization technique used in mass spectrometric analysis of biomolecules. Recent studies have reported the success of this technique in the biomarker discovery for human cancer. To identify novel biomarkers associated with hepatocarcinogenesis, both MALDI-imaging and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were performed on histology-proven HCC and adjacent non-tumor tissues. The mass profile was generated from a total of twenty cases of paired HCC tissues. Using this approach, we found that patients with HBV infection usually have up-regulated peaks at around 5-6 kDa, 11-12 kDa and 13-15 kDa than those with HCV infection (P < 0.05), respectively. Besides, up-regulated peaks at around 8-9 kDa and 15-16 kDa were only detected in moderately differentiated HCC tissue (P < 0.05). The results suggest the existence of potential oncogenes for HCC in these mass ranges of proteins. The gradient SDS-PAGE was also applied to the same tissue pairs, and compared with the results of MALDI-imaging. Finally, in-gel digestion together with tandem mass spectrometry was carried out on candidate masses after Coomassie blue or silver staining. Several interesting genes were identified, including ribonuclease UK114, ubiquitin specific proteases 47 (USP47), progesterone receptor membrane component 1 (PGRMC1) and Galectin-4 (Gal-4). Further investigation on primary tissues found that mRNA expression of UK114 was suppressed in over half of HCCs compared to non-tumor part of liver, consistent with recent report showing markedly reduced expression of UK114 in primary HCC tissues and cancer cell lines. However, higher USP47 mRNA expression in the remaining half of HCCs compared to non-tumor part of liver. So, USP47 might play a role in hepatocarcinogenesis. In addition, expression of PGRMC1 was suppressed in about half of primary HCCs; whereas higher level of PGRMC1 was also revealed in another half of HCCs compared to non-tumor tissue. Interestingly, a comparable expression pattern of PGRMC1 and vascular endothelial growth factor (VEGF) was observed in HCC cell line panel. Given that VEGF is a well known hypoxia-induced angiogenic factor, the possibility of PGRMC1 as a biomarker of hypoxia is being tested in vitro. As for Gal-4, its mRNA expression of was suppressed in about half of HCCs, while the remaining HCCs exhibited the reversed pattern compared with non-tumor tissue. The potential significance of Gal-4 in the differentiation and/or proliferation of HCC will be clarified in the future. In terms of epithelial membrane protein 3 (EMP3), its mRNA expression tends to be higher in late stage HCC cell lines from PLC through SKHep1 cell lines. Functional assays in vitro revealed that transient transfection of EMP3 in HepG2 or HEK293 cells results in reduced cell growth. In addition, overexpression of EMP3 in HEK293 cells inhibits the cell migration. As a result, EMP3 may function as a suppressor gene for HCC.
論文目次 1. Abstract in Chinese...................................Ⅰ
2. Abstract in English...................................Ⅲ
3. Acknowledgements......................................Ⅴ
4. Contents..............................................Ⅵ
5. Index of Tables.......................................Ⅶ
6. Index of Figures......................................Ⅷ
7. Abbreviations.........................................Ⅹ
8. Introduction..........................................1
9. Materials and Methods.................................6
10. Results.............................................14
11. Discussion..........................................22
12. References..........................................27
13. Appendix of Tables..................................32
14. Appendix of Figures.................................59
參考文獻 Acevedo, L. G., et al. Analysis of the mechanisms mediating tumor-specific changes in gene expression in human liver tumors. Cancer Res 68(8), 2641-2651 (2008)
Alaminos, M., et al. EMP3, a myelin-related gene located in the critical 19q13.3 region, is epigenetically silenced and exhibits features of a candidate tumor suppressor in glioma and neuroblastoma. Cancer Res 65(7), 2565-2571 (2005)
Asagi, K., et al. Purification, characterization and differentiation-dependent expression of a perchloric acid soluble protein from rat kidney. Nephron 79, 80-90 (1998)
Barondes, S. H., et al. Galectins. Structure and function of a large family of animal lectins. J. Biol. Chem. 269, 20807–20810 (1994)
Cahill, M. A., et al. Progesterone receptor membrane component 1: an integrative review. J Steroid Biochem Mol Biol 105(1-5), 16-36 (2007)
Chaurand, P., et al. Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections. Curr Opin Chem Biol 6, 676-681 (2002)
Chaurand, P., et al. Imaging mass spectrometry: principles and potentials. Toxicol Pathol 33, 92-101 (2005)
Chen, J.S., et al. Strategies to target HER2/neu overexpression for cancer therapy. Drug Resist Updat 6(3), 129-136 (2003)
Chong, C.L., et al. Decreased expression of UK114 is related to the differentiation status of human hepatocellular carcinoma. Cancer Epidemiol Biomarkers Prev 17(3), 535-542 (2008)
Craven, R. J., et al. PGRMC1: a new biomarker for the estrogen receptor in breast cancer. Breast Cancer Res. 10(6), 113 (2008)
Crudden, G., et al. Overexpression of the cytochrome p450 activator hpr6 (heme-1 domain protein/ human progesterone receptor) in tumors. Tumour Biol 26, 142-146 (2005)
Daviet, L., et al. Targeting ubiquitin specific proteases for drug discovery. Biochimie 90(2), 270-283 (2008)
Deininger, S. O., et al. MALDI imaging combined with hierarchical clustering as a new tool for the interpretation of complex human cancers. J Proteome Res 7(12), 5230-5236 (2008)
El-Serag, H. B., et al. Hepatocellular carcinoma: Epidemiology and Molecular Carcinogenesis. Rev Basic Clin Gastroenterol 132, 2557–2576 (2007)
Farazi, P. A., et al. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 6, 674-687 (2006)
Fuchs, C. S., et al. A phase II trial of gemcitabine in patients with advanced hepatocellular carcinoma. Cancer 94, 3186–3191 (2002)
Fumoto, S., et al. EMP3 as a tumor suppressor gene for esophageal squamous cell carcinoma. Cancer Lett 274(1), 25-32 (2009)
Hanash, S. Integrated global profiling of cancer. Nat Rev Cancer 4, 638-644 (2004)
Hernandez, J. D., et al. Ah, sweet mystery of death! Galectins and control of cell fate. Glycobiology 12, 127R–136R (2002)
Herring, K. D., et al. Direct tissue analysis by matrix-assisted laser desorption ionization mass spectrometry: application to kidney biology. Semin Nephrol 27, 597- 608 (2007)
Huang, J., et al. Correlation between genomic DNA copy number alterations and transcriptional expression in hepatitis B virus-associated hepatocellular carcinoma. FEBS Lett 580(15), 3571-3581 (2006)
Hughes, R. C. Secretion of the galectin family of mammalian carbohydrate-binding proteins. Biochim. Biophys. Acta 1473, 172–185 (1999)
Johnson, P. J., et al. The role of serum a-fetoprotein estimation in the diagnosis and management of hepatocellular carcinoma. Clin LiverDis 5, 145-159 (2001)
Kaneki, K., et al. Purification, characterization and developmental expression of pig liver PSP. Comp Biochem Physiol B Biochem Mol Biol 134, 571-578 (2003)
Katoh, H., et al. Genetic profile of hepatocellular carcinoma revealed by array-based comparative genomic hybridization: identification of genetic indicators to predict patient outcome. J Hepatol 43(5), 863-874 (2005)
Kondoh, N., et al. Identification and characterization of genes associated with human hepatocellular carcinogenesis. Cancer Res 59(19), 4990-4996 (1999)
Lopez, P. M., et al. Systematic review: evidence-based management of hepatocellular carcinoma: an updated analysis of randomized controlled trials. Aliment Pharmacol Ther 23, 1535–1547 (2006)
Migliore, C., et al. Molecular cancer therapy: can our expectation be MET? Eur J Cancer 44(5), 641-651 (2008)
Muller, C. Hepatocellular carcinoma—rising incidence, changing therapeutic
strategies. Wien Med Wochenschr 156, 404–409 (2006)
Nalepa, G., et al. Drug discovery in the ubiquitin-proteasome system. Nat Rev Drug 5(7), 596-613 (2006)
Neubauer, H., et al. Breast cancer proteomics reveals correlation between estrogen receptor status and differential phosphorylation of PGRMC1. Breast Cancer Res. 5, R85 (2008)
Nishimura, T., et al. Genome-wide semiquantitative microsatellite analysis of human hepatocellular carcinoma: discrete mapping of smallest region of overlap of recurrent chromosomal gains and losses. Cancer Genet Cytogenet 167(1), 57-65 (2006)
Nordin, H., et al. Purification, characterization and developmental expression of chick (Gallus domesticus) liver PSP protein. Comp Biochem Physiol B Biochem Mol Biol 128, 135-143 (2001)
Oda, Y., et al. Soluble lactose-binding lectin from rat intestine with two different carbohydrate-binding domains in the same peptide chain. J. Biol. Chem. 268, 5929–5939 (1993)
Pang, R., et al. Molecular pathways in hepatocellular carcinoma. Cancer Lett 242, 151–167 (2006)
Peluso J. J., et al. Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1. J Clin Endocrinol Metab 93, 1592-1599 (2008)
Poon, R. T., et al. Improving survival results after resection of hepatocellular carcinoma: a prospective study of 377 patients over 10 years. Ann Surg 234, 63–70 (2001)
Poon, R. T., et al. Locoregional therapies for hepatocellular carcinoma: a critical review from the surgeon’s perspective. Ann Surg 235, 466–486 (2002)
Poon, R. T., et al. Resection prior to liver transplantation for hepatocellular carcinoma: a strategy of optimizing the role of resection and transplantation in cirrhotic patients with preserved liver function. Liver Transpl 10, 813–815 (2004)
Rechreche, H., et al. Cloning and expression of the mRNA of human galectin-4, an S-type lectin down-regulated in colorectal cancer. Eur. J. Biochem. 248, 225–230 (1997)
Santamaría, E., et al. Toward the discovery of new biomarkers of hepatocellular carcinoma by proteomics. Liver Int 27(2), 163-73 (2007)
Schlaeger, C., et al. Etiology-dependent molecular mechanisms in human hepatocarcinogenesis. Hepatology 47(2), 511-520 (2008)
Singhal, S., et al. Deubiquitylating enzymes and disease. BMC Biochem 9, Suppl 1, S3 (2008)
Stauber, J., et al. MALDI imaging of formalin-fixed paraffin-embedded tissues: application to model animals of Parkinson disease for biomarker hunting. J Proteome Res 7(3), 969-978 (2008)
Suzuki, K., et al. Purification, characterization and developmental expression of rat brain PSP protein. Biochim Biophys Acta 1527, 47-53 (2001)
Taketa, K. a-Fetoprotein: reevaluation in hepatology. Hepatology 12, 1420-1432 (1990)
Taylor, V., et al. Epithelial membrane protein-2 and epithelial membrane protein-3: two novel members of the peripheral myelin protein 22 gene family. Gene 175(1-2), 115-120 (1996)
Thomas, M. B. Systemic therapy for hepatocellular carcinoma. The Cancer Journal 14(2), 123-127 (2008)
Thomas, R. M., et al. The RON receptor tyrosine kinase mediates oncogenic phenotypes in pancreatic cancer cells and is increasingly expressed during pancreatic cancer progression. Cancer Res 67(13), 6075-6082 (2007)
Tyers, M., et al. From genomics to proteomics. Nature 422(6928), 193-197 (2003)
Weihua, Z., et al. Survival of cancer cells is maintained by EGFR independent of its kinase activity. Cancer cell 13(5), 385-393 (2008)
Yeo, W. A randomized phase III study of doxorubicin versus cisplatin/interferon alpha-2b/doxorubicin/fluorouracil (PIAF) combination chemotherapy for unresectable
hepatocellular carcinoma. J Natl Cancer Inst 97, 1532–2538 (2005)
Zhou, X. D., et al. Experience of 1000 patients who underwent hepatectomy for small hepatocellular carcinoma. Cancer 91, 1479-1486 (2001)
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