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研究生：	吳宗樺 Wu, Tzong-huah
論文名稱：	研究一個具有腫瘤胚胎表現型的功能未知基因 The study of an unknown function gene with oncofetal-like expression pattern
指導教授：	何中良 Ho, Chung-liang 蔣輯武 Chiang, Chi-wu
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：	2007
畢業學年度：	95
語文別：	中文
論文頁數：	94
中文關鍵詞：	腫瘤胚基因
外文關鍵詞：	overlap PCR, LRR domain, Leucine-rich repeat, oncofetal gene, cancer
相關次數：	點閱：121 下載：3
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正常狀態下，胚胎發育過程中參與的基因在出生後便不再表現，可是當腫瘤生成過程中可能會再度被啟動，這類的基因有人稱它為腫瘤胚﹙Oncofetal﹚基因。在臨床應用上，這類的基因是很好的診斷指標，藉由抽血偵測此類的指標基因即可早期發現腫瘤的形成，甚至在腫瘤形成前及早預防。而本實驗室基於如此的概念之下，從生物資訊龐大的EST﹙Expressed Sequence Taq, 表現序列標幟﹚資料庫中，找出有可能的腫瘤胚基因，之後藉RT-PCR的方式篩選出具有腫瘤胚表現型的基因進行研究。本篇論文研究的主題EGRT-U5﹙Embryonic Gene Re-express in Tumor-Unknown 5﹚是一個已知序列但生物功能未知的基因，PCR篩選結果發現它幾乎在所有被測腫瘤細胞株中都會表現，可是在正常組織中，僅有某些器官﹙例如：胎兒時期的肝與腦、正常的腦、骨髓、男性生殖腺﹚表現量比較多，其餘器官雖然有但表現明顯減少，而週邊血也幾乎檢測不到。在臨床檢體腫瘤非腫瘤配對檢測結果顯示此基因具有類似腫瘤胚基因的表現型。根據生物資訊資料庫的預測，此基因含有數個Leucine-rich repeat ﹙LRR﹚ domain，此LRR domain在先前的報導中有提到：含有LRR domain的蛋白質可能參與哺乳類早期的胚胎發育、神經發育、細胞極化或是調控基因的表達，也可能參與細胞凋亡的訊息傳遞過程。這些現象與EGRT-U5的狀態有些類似。本人首先用了各種方式終於把這個基因的全長送進可以表達的質體中，接著再製造出3種不同的truncated forms。把這些constructs送進293T及BHK腫瘤細胞株過量表現，從西方墨點法﹙Western blotting﹚的結果顯示此基因產生的蛋白質處於細胞裡的不溶狀態，必需用尿酸﹙Urea﹚才能將它溶解出來，再加上以生物資訊的方法預測出幾個可能跟actin結合的相關結構，因此推測此蛋白質很可能在生物功能上扮演著focal adhesion相關或是調節細胞骨架的角色。而在螢光染色結果顯示在細胞膜上、細胞核中都有發現此基因的蹤跡，另外於細胞貼於蓋玻片的貼面上觸手的輪廓會特別明顯，細胞核周圍會形成一顆顆點狀聚集，但是卻無法跟vincullin的位置重合﹙colocalize﹚。當此蛋白質缺乏LRR domains時，不難發現大部分的蛋白質跑進細胞核中。而在缺乏末端379個胺基酸的狀態下，大致上與全長相符，唯原本可以看到螢光在細胞突出部的現象消失了。以上種種新發現的現象發生的原因仍有待更深入研究證實。

Some researchers believe that the process of tumorigenesis represent a disorganized organ genesis in adult life, which tries but fails to replay the process of embryo development. Oncofetal proteins, such as α-fetoprotein﹙AFP﹚, are proteins that are expressed in embryonic stage, down-regulated after birth, but re-expressed in tumors. These proteins are useful in cancer diagnoses and follow-up. Our laboratory used a bioinformatic approach to find a gene with oncofetal-like expression patterns through screening for their expression in tumor cell lines, peripheral blood samples, non-tumor tissues, and tumor tissues, which was designated “Embryonic Gene Re-express in Tumor-Unknown 5﹙EGRT-U5﹚”. By sequence prediction, this gene encodes a 1372 a.a., ~150kD protein with several Leucine-rich repeat ﹙LRR﹚ domains, which in general are believed to be important in protein-protein interaction. The study is to investigate the functions of EGRT-U5 through transient overexpression in tumor cell lines. I have cloned full length EGRT-U5 into 3 reporter vectors ﹙pEGFP-C1, pEGFP-N1 and pcDNA3.1/V5-His﹚ and created 3 different truncated eGFP fusion proteins. The fluorescents of those constructs shows that the localization of EGRT-U5 GFP fusion proteins may be accumulating on cell membranes, aggregating insolubly in middle of cells , or recruited into nucleus. The bioinformatics prediction and western blot imply that EGRT-U5 would strongly associate to cytoskeleton through several predicted actin binding domains and to other related cytoskeleton associating complexes through LRR domains. Moreover, LRR proteins were reported as proteins participate in many biologically important processes, such as hormone–receptor interactions, enzyme inhibition, cell adhesion and cellular trafficking. Many My study reveals that the function of EGRT-U5 would involved in cell adhesion and trafficking and change cell morphology through regulating dynamics of cytoskeleton

目錄
中文摘要	I
英文摘要	II
誌謝	III
目錄	V
表目錄	VIII
圖目錄	VIII

正文
1 緒論﹙Introduction﹚	p.1
1.1 Expressed Sequence Tags資料庫	p.1
1.2 Oncofetal Protein	p.2
1.3 找尋新的oncofetal gene–EGRT-U5	p.2
1.4 Bioinformatics	p.4
1.4.1 National Center for Biotechnology Information﹙NCBI﹚	p.4
1.4.2 Ensembl	p.5
1.4.3 SMART﹙Simple Molecular Architecture Research Tool﹚	p.5
1.4.4 WoLF PSORT	p.5
1.5 EGRT-U5 Full-length Clone	p.6
2 材料與方法﹙Material & Method﹚	p.7
2.1 EGRT-U5 Full-length cloning	p.7
2.1.1 Human fetal brain total RNA and BC008134 clone	p.7
2.1.2 RT-PCR	p.7
2.1.3 Overlap PCR	p.8
2.1.4 DNA purification	p.8
2.1.5 Calculate DNA concentration	p.8
2.1.6 TA cloning	p.9
2.1.7 Directional cloning	p.9
2.1.8 Sequencing	p.10
2.2 Gene overexpression	p.10
2.2.1 Cell lines and cell culture	p.10
2.2.2 Transfection	p.10
2.3 Western blotting procedure	p.10
2.3.1 Sample preparation	p.10
2.3.2Western blotting	p.11
2.4 Bioinfomatics	p.12
2.5 Fluorescent Microscopy	p.12
2.5.1 Sample preparation	p.12
2.5.2 Fluorescent Microscopy	p.13
3 結果﹙Results﹚	p.14
3.1 EGRT-U5 Full-length Clone	p.14
3.1.1 EGRT-U5 Full-length Cloning 原始設計	p.14
3.1.2測試出可行的primer pairs	p.14
3.1.3 解決mutation問題	p.15
3.1.4 將各個小片段組合成EGRT-U5 full-length clone	p.16
3.2 Functional Assay	p.18
3.2.1 Western Blotting顯示EGRT-U5為一insoluble蛋白質	p.18
3.2.2 Fluorescent Microscopy	p.19
4 討論﹙Discussion﹚	p.23
4.1 EGRT-U5 full-length cloning	p.23
4.2 Western blotting	p.25
4.3 Fluorescent Microscopy	p.26
4.4 LRR protein	p.27
5 參考文獻﹙Reference﹚	p.71
6 附錄﹙Appendixes﹚	p.74
 
表目錄
表一、利用SMART軟體預測出EGRT-U5可信的domains	p.29
表二、利用SMART軟體預測出EGRT-U5 less significant domains	p.29
表三、利用WoLF PSORT預測EGRT-U5可能的subcellular localization	p.34
表四、PCR反應之組成與condition	p.35
表五、EGRT-U5原始設計的Primer Sequences for full-length cloning	p.36
表六、EGRT-U5 clone出全長的Primer Sequences	p.36
表七、本研究所有constructs與Insert DNA連結位置附近的序列	p.37
表八、運用於functional assay中所有constructs的基本物理化學性質	p.40

圖目錄
圖一、EGRT-U5在各種tumor cell lines、normal cell lines及peripheral blood samples中表現的情形	p.41
圖二、EGRT-U5在各個器官的腫瘤–非腫瘤配對中表現的情形	p.42
圖三、Rat EGRT-U5 homologue在胚胎、剛出生﹙P0﹚及20週成鼠中表現的情形	p.43
圖四、利用Bioinformatic工具找到的EGRT-U5的資訊	p.44
圖五、EGRT-U5 full-length cloning過程示意圖	p.45
圖六、EGRT-U5 full-length cloning初始設計及最終可行之primer pairs示意圖	p.46
圖七、測U5A-FR最低的PCR cycle數	p.47
圖八、EGRT-U5 full-length clone與NM_138360.2序列比對結果	p.48
圖九、EGRT-U5各片段及全長constructs示意圖	p.49
圖十、使用於EGRT-U5 functional assay中的constructs	p.50
圖十一、本篇研究中所有使用的載體圖譜	p.51
圖十二、EGRT-U5 western blotting結果	p.52
圖十三、U5全長於293T細胞中不同層的表現情形及U5一些代表性結構	p.53
圖十四、eGFP-N1表現於BHK細胞的40X螢光圖	p.55
圖十五、eGFP-N1表現於BHK細胞的100X螢光圖	p.55
圖十六、U5-Full-C1表現於BHK細胞的40X螢光圖	p.55
圖十七、U5-Full-C1表現於BHK細胞的100X螢光圖	p.57
圖十八、U5-Full-N1表現於BHK細胞的40X螢光圖	p.57
圖十九、U5-Full-N1表現於BHK細胞的100X螢光圖	p.57
圖二十、U5-Full-N1表現於BHK細胞的100X螢光圖II	p.59
圖二十一、U5-1,2-C1表現於BHK細胞的40X螢光圖	p.59
圖二十二、U5-1,2-C1表現於BHK細胞的100X螢光圖I	p.59
圖二十三、U5-1,2-C1表現於BHK細胞的100X螢光圖II	p.61
圖二十四、U5-2,3-C3表現於BHK細胞的40X螢光圖	p.61
圖二十五、U5-2,3-C3表現於BHK細胞的100X螢光圖	p.61
圖二十六、U5-3-N1表現於BHK細胞的40X螢光圖	p.63
圖二十七、U5-3-N1表現於BHK細胞的100X螢光圖	p.63
圖二十八、Virus U5-Full-N1表現於BHK細胞的40X螢光圖	p.63
圖二十九、Virus U5-Full-N1表現於BHK細胞的100X螢光圖I	p.65
圖三十、Virus U5-Full-N1表現於BHK細胞的100X螢光圖II	p.65
圖三十一、表現於BHK及293T細胞的U5-Full-N1無法與vincullin colocalize	p.67
圖三十二、1kb clone產生1個point mutation的情況下mutation rate與PCR cycle數之間的關係	p.69
圖三十三、解決Overlap PCR的過程示意圖	p.70
                                    

Bostjan Kobe and Andrey V Kajava, The Leucine-rich repeat as a protein recognition motif, Curr Opin Struct Biol, 11(6):725-32, 2001 Dec.
Danielle and Jean Thierry-Mieg, Michel Potdevin, Mark Sienkiewicz, Vahan Simonyan.The AceView genes. www.ncbi.nlm.nih.gov/IEB/Research/Acembly AceView: Identification and functional annotation of cDNA-supported genes in eukaryotes.
Danielle Thierry-Mieg and Jean Thierry-Mieg, AceView: a comprehensive cDNA-supported gene and transcripts annotation, Genome Biology 2006, 7(Suppl 1):S12.
Bruce A. White, PCR Protocols – current methods and applications, Ch25, pp251-260, 1993.
Entrez Gene : C14orf121, NCBI, http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=full_report&list_uids=90668, Searching Date : 1st, July, 2007.
Entrez Conserved Domains : C14orf121, NCBI, http://www.ncbi.nlm.nih.gov/sites/entrez?itool=gene_full_report&DbFrom=gene&Cmd=
Link&LinkName=gene_cdd&IdsFromResult=90668, Searching Date : 1st, July, 2007.
Ivica Letunic, Richard R. Copley1, Birgit Pils2, Stefan Pinkert2, Jörg Schultz2 and Peer Bork, SMART 5: domains in the context of genomes and networks, Nucleic Acids Research, Vol. 34, 2006
Jörg Schultz, Frank Milpetz, Peer Bork, and Chris P. Ponting, SMART, a simple modular architecture research tool: Identification of signaling domains, Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 5857–5864, May 1998.
Joseph Sambrook and David W. Russell, Molecular cloning – A laboratory manual, Cold spring Harbor Laboratory, Vol. 2, Ch8, p.87, 2001.
Marchler-Bauer A, Anderson JB, Cherukuri PF, DeWeese-Scott C, Geer LY, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Liebert CA, Liu C, Lu F, Marchler GH, Mullokandov M, Shoemaker BA, Simonyan V, Song JS, Thiessen PA, Yamashita RA, Yin JJ, Bryant SH, CDD: a Conserved Domain Database for protein classification. Nucleic Acids Res. 2005;33 Database Issue:D192-6.
Marilyn Monk and Cathy Holding, Human-embryonic genes re-expressed in cancer cells, Oncogene 20, 8085-8091, 2001.
NCBI AceView: C14orf121, Yuji Kohara and Tsodas Shin-i, http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/
av.cgi?c=geneid&org=9606&l=90668, Searching Date : 1st, July, 2007.
Paul Horton, Keun-Joon Park, Takeshi Obayashi, Naoya Fujita, Hajime Harada, C.J. Adams-Collier, & Kenta Nakai, "WoLF PSORT: Protein Localization Predictor", Nucleic Acids Research, doi:10.1093/nar/gkm259, 2007.
Paul Horton, Keun-Joon Park, Takeshi Obayashi & Kenta Nakai, "Protein Subcellular Localization Prediction with WoLF PSORT", Proceedings of the 4th Annual Asia Pacific Bioinformatics Conference APBC06, Taipei, Taiwan. pp. 39-48, 2006.
Peter Alexander, Fetal “antigens” in Cancer, Nature Vol. 235 January 21, 1972.
Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Church DM, DiCuccio M, Edgar R, Federhen S, Helmberg W, Kenton DL, Khovayko O, Lipman DJ, Madden TL, Maglott DR, Ostell J, Pontius JU, Pruitt KD, Schuler GD, Schriml LM, Sequeira E, Sherry ST, Sirotkin K, Starchenko G, Suzek TO, Tatusov R, Tatusova TA, Wagner L, Yaschenko E, Database resources of the National Center for Biotechnology Information, Nucleic Acids Res., Jan, 2005. (NCBI)
T. J. P. Hubbard, B. L. Aken, K. Beal1, B. Ballester1, M. Caccamo, Y. Chen, L. Clarke, G. Coates, F. Cunningham, T. Cutts, T. Down, S. C. Dyer, S. Fitzgerald, J. Fernandez-Banet, S. Graf, S. Haider, M. Hammond, J. Herrero, R. Holland, K. Howe, K. Howe, N. Johnson, A. Kahari, D. Keefe, F. Kokocinski, E. Kulesha, D. Lawson, I. Longden, C. Melsopp, K. Megy, P. Meidl, B. Overduin, A. Parker, A. Prlic, S. Rice, D. Rios, M. Schuster, I. Sealy, J. Severin, G. Slater, D. Smedley, G. Spudich, S. Trevanion, A. Vilella, J. Vogel, S. White, M. Wood, T. Cox, V. Curwen, R. Durbin, X. M. Fernandez-Suarez, P. Flicek, A. Kasprzyk, G. Proctor, S. Searle, J. Smith, A. Ureta-Vidal and E. Birney, Ensembl 2007, Nucleic Acids Res. 2007 Jan 1.
周靖恆，結合生物資訊的方法探討腫瘤相關基因在人類腎臟癌中的表現，國立成功大學分子醫學研究所碩士論文，民國九十四年。

校內：2010-08-30公開
校外：2012-08-30公開

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