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研究生：	徐一尹 Hsu, Yi-Yin
論文名稱：	研究ENO1基因的產物在頭頸癌細胞中扮演的角色 The study of ENO1 gene products in head and neck cancer cells
指導教授：	吳梨華 Wu, Li-Wha
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2009
畢業學年度：	98
語文別：	英文
論文頁數：	44
中文關鍵詞：	烯醇化酶
外文關鍵詞：	ENO1
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醣解基因ENO1 可經由轉譯作出烯醇化酶(Enolase 1)，其可催化2-磷酸甘油酸(2-phospho-D-glycerate)轉換成磷酸烯醇丙酮(phosphoenolpyruvate)。此外，ENO1基因被推測可經由替代式轉譯的方式，利用抵enolase 1上第97個甲硫胺酸做出另一核內蛋白質 MBP1。在我們先前的研究發現，ENO1的過度表現不僅在口腔癌病人癒後為一負向因子其也增加細胞的轉形。 ENO1 knockdown 降低了細胞轉形的狀況，例如：細胞生長，這顯示了ENO1擁有促進腫瘤形成的角色。然而，與ENO1 knockdown所調控生長抑制相關的機制，目前仍不清楚。首先，我們利用測試細胞生長及利用流式細胞分析法，確認ENO1 knockdown對細胞生長，細胞週期及細胞凋亡所造成的影響。我們的研究發現，ENO1 knockdown 可經由將細胞拘留在G1階段的方式造成細胞的生長顯著降低和細胞凋零。利用DNA微陣列的分析去比較控制組及ENO1 knockdown的CAL27細胞，發現經由ENO1 knockdown，可誘發p21 mRNA 的表現量。之後，我們再利用半定量RT-PCR確認ENO1 knockdown所誘發的p21 mRNA 表現。在CAL27及KYSE170這兩株頭頸癌的細胞株中，比較p21 mRNA及蛋白質的表現，發現p21在轉錄及後轉錄的階段有不同的調控。從經由actinomycin D 或 cycloheximide的knockdown 細胞中分離出蛋白質，並做西方墨點法分析，結果顯示由ENO1 knockdown所誘發的p21表現可在轉錄階段或後轉錄階段或兩者同時進行調控。為了測試p21在ENO1 knockdown 抑制細胞進行過程中所參與的角色，我們利用測試細胞的生長來研究knock down p21在ENO1 knockdown的細胞中所在造成的影響。Knockdown p21的表現削減了ENO1 knockdown影響細胞生長的能力，這暗示了p21在ENO1 knockdown的細胞中為一正向調控因子。而在一些頭頸癌細胞株株中，也偵測到enoalse 及p21的表現量呈現相反的關係。為了區別兩個同為ENO1基因產物，enoalse 1及MBP1，對細胞生長的影響，我們利用定點突變將黃色螢光蛋白融合不同的ENO1突變體，分別為Eno1mut1 (只產生MBP1)， Eno1mut2 (只產生 enolase 1)，Eno1mut 3 (只產生 enolase 1) 和 MBP1。由西方墨點法的分析，我們確認ENO1 主要表現enoalse 1次要表現MBP1，這種差異性的表現是利用天生的轉譯起始密碼所造成。而我們也發現，YFP-ENO1這個主要表enolase 1的融合蛋白顯著的增加細胞生長，這樣的結果和enolase 1在人類癌症中多過度表現的狀況是一致的。雖然MBP1是由ENO1基因所做出，且被認為會抑制細胞的生長，但MBP1的低表現量使其對降低細胞生長沒有影響，這或許可以解釋為什麼ENO1常被當作生長促進基因而不是抑制生長的基因。總而言之，醣解基因ENO1可能在控管細胞週期中扮演其他的角色。ENO1及其下游的目標基因值得更深入的探討，使其未來得以在頭頸癌中用來當作治療或診斷的標的。

ENO1 encodes a glycolytic enolase 1, catalyzing the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate. Besides, ENO1 also encodes a nuclear MYC-binding protein 1 (MBP1) presumably via alternative initiation at Met97 from one single transcript. In our previous studies, we found that ENO1 overexpression is not only a poor prognostic factor for oral cancer patient survival but also promotes cellular transformation. ENO1 knockdown reduced the stimulatory effect on cell transformation including cell growth, suggesting a tumor promoting role of ENO1. However, the mechanism responsible for ENO1 knockdown-mediated growth inhibition remains unknown. We first confirmed the effect of ENO1 knockdown on cell proliferation, cell cycle progression and apoptosis using cell proliferation and flow cytometry. We found that ENO1 knockdown significantly reduced cell proliferation via increased G1 arrest and apoptosis. By comparing the gene expression profiles of vector control and ENO1 knockdown CAL-27 oral cancer cells using DNA microarray analysis, the mRNA expression of cyclin-dependent kinase inhibitor, p21, was most induced by ENO1 knockdown. ENO1 knockdown-induced p21 mRNA expression was later confirmed by semi-quantitative RT-PCR. When compared the mRNA with protein expression of p21 in two head and neck cancer lines, CAL27 and KYSE170, we found a differential regulation of p21 at transcriptional or post-transcriptional levels. Western blot analysis of protein lysates isolated from knockdown cells treated with actinomycin D or cycloheximide indicates the induction of p21 by ENO1 knockdown can be modulated at transcriptional, post-transcriptional or both. To study the involvement of p21 in the ENO1-knockdown mediated inhibition of cell cycle progression, cell doubling was used to study the effect of p21 knockdown in ENO1-knockdown cells. Knocking down the expression of p21 attenuates the ability of ENO1-knockdown to proliferate, indicating a positive role of p21 in ENO1 knockdown. The inverse expression of enolase 1 and p21 was also detected in several head and neck cancer cells. To differentiate the effect of both ENO1 gene products, enolase 1 and MBP1 on cell proliferation, we fused different ENO1 mutants, ENO1mut1 (encoding MPB only), ENO1mut2 (encoding enolase 1 only), ENO1mut 3 (encoding enolase 1 only) and MBP1, with yellow fluorescent protein (YFP) using site-directed mutagenesis. Western blot analysis confirmed a differential expression of major enolase 1 and minor MBP1 using native translation initiation codons. Consistent with frequent overexpression of enolase 1 in human cancer, YFP-ENO1 fused constructs expressing predominant enolase 1 significantly increased cell proliferation. Although MBP1 encoded by ENO1 was reported to suppress cell growth, the inability of low-expressing MBP1 to reduce cell growth might explain why ENO1 often behaves as a growth promoting gene rather than suppressing gene. Together, glycolytic ENO1 may have an additional role in holding cell cycle in check. The studies of ENO1 and its downstream targets might potentially be explored as potential therapeutic or prognostic targets for treating head and neck cancer.

I. Abstract in Chinese..........................Ⅱ 
II. Abstract in English.........................Ⅳ 
III. Acknowledgement..........................Ⅵ 
IV. Table of content...........................Ⅶ 
V. List of figures............................Ⅸ 
VI. Abbreviations............................Ⅹ 

VII. Introduction 
1.1 Head and neck cancer........................1 
1.2 Glycolysis............................1 
1.3 Glycolysis and cancer.........................2 
1.4 Enolse..............................3 
1.5 ENO1 gene products.........................4 
VIII. Hypothesis...............................5 
IX. Specific aim...............................5 
X. Materials and methods 
2.1 Materials............................6 
2.2 Cell culture............................7 
2.3 Plasmid constructs........................8 
2.4 Transient transfection.........................8 
2.5 shRNA knockdown.........................8 
2.6 Semi-quantitative RT-PCR.......................9 
2.7 Site-directed mutagenesis.......................9 
2.8 Western blotting..........................10 
2.9 Fluorescence microscopy......................10 
2.10 Cell proliferation and doubling.....................11 
2.11 Cell cycle analysis using double thymidine block............11 
2.12 Apoptosis a nalysis..........................11 
2.13 Statistical a nalysis..........................12 
XI. Results 
3.1 ENO1 knockdown decreased cell proliferation via induced G1 arrest or apoptosis.............................13 
3.2 Induced expression of p21 by ENO1 knockdown..........14 
3.3 The inverse expression of enolase 1 and p21 in head and neck cancer cells....14 
3.4 Etopic alteration of p21 expression changed the cell proliferation induced by ENO1 overexpression.........................15 
3.5 ENO1 knockdown induced p21 expression both at transcriptional and post- transcriptional level........................16 
3.6 The involvement of various kinases in ENO1-induced p21 expression.....17 
3.7 Subcellular localization of enolase 1 and MBP1 in transiently transfect 293T cells..............................17 
3.8 Differential effects of enolase 1 and MBP1 on cell proliferation.......18 
XII. Discussion...............................20 
XIII. Reference..............................22
                                    

1. Psyrri, A., et al., Head and neck cancer. J Oncol, 2009. 2009: p. 358098.

2. Million RR, C.N., Clark JR, Cancer of the head and neck., in Cancer, principles & practice of oncology, H.S. DeVita VT, Rosenberg SA, Editor. 1989, JB Lippincott Company: Philadelphia(PA). p. 488–590.

3. Cooper, J.S., et al., Second malignancies in patients who have head and neck cancer: incidence, effect on survival and implications based on the RTOG experience. Int J Radiat Oncol Biol Phys, 1989. 17(3): p. 449-56.

4. McDonald, S., et al., Second malignant tumors in patients with laryngeal carcinoma: diagnosis, treatment, and prevention. Int J Radiat Oncol Biol Phys, 1989. 17(3): p. 457-65.

5. Rodriguez, T., et al., Risk factors for oral and pharyngeal cancer in young adults. Oral Oncol, 2004. 40(2): p. 207-13.

6. Silverman, S., Jr., M. Gorsky, and F. Lozada, Oral leukoplakia and malignant transformation. A follow-up study of 257 patients. Cancer, 1984. 53(3): p. 563-8.

7. Pelicano, H., et al., Glycolysis inhibition for anticancer treatment. Oncogene, 2006. 25(34): p. 4633-46.

8. Herrero, P., et al., Transcriptional regulation of the Saccharomyces cerevisiae HXK1, HXK2 and GLK1 genes. Yeast, 1995. 11(2): p. 137-44.

9. Feo, S., et al., ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1). FEBS letters, 2000. 473(1): p. 47-52.

10. Rodriguez, A., et al., The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae. Biochem J, 2001. 355(Pt 3): p. 625-31.

11. Zheng, L., R.G. Roeder, and Y. Luo, S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell, 2003. 114(2): p. 255-66.

12. Niederacher, D. and K.D. Entian, Characterization of Hex2 protein, a negative regulatory element necessary for glucose repression in yeast. Eur J Biochem, 1991. 200(2): p. 311-9.

13. Gatenby, R.A. and R.J. Gillies, Why do cancers have high aerobic glycolysis? Nat Rev Cancer, 2004. 4(11): p. 891-9.

14. O., W., The Metabolism of Tumors. 1930, London.

15. Warburg, O., On the origin of cancer cells. Science, 1956. 123(3191): p. 309-14.

16. Pearce, J.M., Y.H. Edwards, and H. Harris, Human enolase isozymes: electrophoretic and biochemical evidence for three loci. Ann Hum Genet, 1976. 39(3): p. 263-76.

17. Racz, A., et al., Gene amplification at chromosome 1pter-p33 including the genes PAX7 and ENO1 in squamous cell lung carcinoma. International journal of oncology, 2000. 17(1): p. 67.

18. Marangos, P.J., A.M. Parma, and F.K. Goodwin, Functional properties of neuronal and glial isoenzymes of brain enolase. J Neurochem, 1978. 31(3): p. 727-32.

19. Li, C., et al., Proteome analysis of human lung squamous carcinoma. Proteomics, 2006. 6(2): p. 547-58.

20. Wold, F., The Enzymes. Vol. 5. 1971, New York: Boyer.

21. Miles, L., et al., Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor. Biochemistry, 1991. 30(6): p. 1682.

22. Dano, K., et al., Plasminogen activation and cancer. Thromb Haemost, 2005. 93(4): p. 676-681.

23. Wygrecka, M., et al., Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung. Blood, 2009. 113(22): p. 5588.

24. Ray, R. and D. Miller, Cloning and characterization of a human c-myc promoter-binding protein. Molecular and cellular biology, 1991. 11(4): p. 2154.

25. Bentley, D. and M. Groudine, A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells. 1986.

26. Subramanian, A. and D.M. Miller, Structural analysis of alpha-enolase. Mapping the functional domains involved in down-regulation of the c-myc protooncogene. J Biol Chem, 2000. 275(8): p. 5958-65.

27. Ray, R.B., et al., Human breast carcinoma cells transfected with the gene encoding a c-myc promoter-binding protein (MBP-1) inhibits tumors in nude mice. Cancer Res, 1995. 55(17): p. 3747-51.

28. Wu, W., et al., Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry. Clin Exp Metastasis, 2002. 19(4): p. 319-26.

29. Lin, S.C., et al., Establishment of OC3 oral carcinoma cell line and identification of NF-kappa B activation responses to areca nut extract. J Oral Pathol Med, 2004. 33(2): p. 79-86.

30. Tsai, S.T., et al., ENO1, a potential prognostic head and neck cancer marker, promotes transformation partly via chemokine CCL20 induction. Eur J Cancer, 2010.

31. Satyanarayana, A., M.B. Hilton, and P. Kaldis, p21 Inhibits Cdk1 in the absence of Cdk2 to maintain the G1/S phase DNA damage checkpoint. Mol Biol Cell, 2008. 19(1): p. 65-77.

32. Sobell, H.M., Actinomycin and DNA transcription. Proc Natl Acad Sci U S A, 1985. 82(16): p. 5328-31.

33. Ennis, H.L. and M. Lubin, CYCLOHEXIMIDE: ASPECTS OF INHIBITION OF PROTEIN SYNTHESIS IN MAMMALIAN CELLS. Science, 1964. 146: p. 1474-6.

34. Sedoris, K.C., S.D. Thomas, and D.M. Miller, Hypoxia induces differential translation of enolase/MBP-1. BMC Cancer, 2010. 10(1): p. 157.

校內：2020-12-30公開
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