Structural, biochemical and clinical analyses of a cancer-related protein, C35 (C17orf37)

癌症相關分子的發現，對於疾病的診斷與治療具有很大的應用潛力。C35 (C17orf37)是一個位於人類第十七號染色體(17q12-21)的新癌症相關基因。由於在前人利用免疫組織染色法（immunohistochemical analysis, IHC）所做的研究中指出，C35蛋白可大量且頻繁地在乳癌病理組織中被偵測到，因此在本實驗中，我們將從結構的分析與對細胞功能的影響，來探討C35蛋白在癌症生成中所扮演的角色。利用反轉錄作用從人類乳癌細胞—SK-BR-3所獲得的C35全長基因，在經過大腸桿菌（E. coli strain BL21）的表現後，我們將所純化的蛋白利用圓二色光譜儀（circular dichroism）和核磁共振儀（nuclear magnetic resonance）分析其結構。從圓二色光譜儀的分析結果顯示，C35的二級結構主要以α-螺旋（α-helix）的形式存在；此外，C35蛋白具有很好的熱耐受性和酸鹼穩定性。在利用核磁共振儀所做的測量中，首先我們利用一維分析法（one-dimensional NMR analysis）先確認C35具有穩定的蛋白構型，接著利用二維法（two-dimensional NMR analysis）對其三級結構做進一步地分析，最後，我們發現C35蛋白具有和硫氧還蛋白相似的構型（thioredoxin-like structure）。為了進一步瞭解C35蛋白和癌症的相關性，在接下來的研究中，我們利用免疫組織染色法，證實了C35也會在胃癌中表現；在反轉錄聚合酵素連鎖反應（RT-PCR）的偵測中，發現C35 mRNA 的表現普遍存在於所有的細胞中；然而在蛋白質的表現上，利用西方點墨法（Western blotting）偵測，我們只在人類乳癌細胞—BT474中，發現內生性C35蛋白的存在。為了探討C35蛋白在正常組織中所扮演的角色，我們利用10種不同的老鼠組織來做內生性C35蛋白的偵測。由於在這些正常組織中，都無法偵測到C35蛋白的存在，因此我們推測C35蛋白並不會在正常組織中表現。最後，我們利用Boyden chamber assay探討C35蛋白對細胞（CHO-K1）功能的影響，實驗結果顯示，C35的大量表現具有促進細胞移動的能力。藉由C35蛋白的初步研究，顯示其與癌症的發生確實有相關性，然而其在癌化過程中所扮演的角色，仍有待更進一步的研究。Identification of cancer-specific biomarkers has enormous potential to enhance detection, treatment, and prognosis. C35 (C17orf37) is a novel gene, which locates at chromosome 17q12-21 and is found to be up-regulated in many cancers. In a previous study, immunohistochemical analysis detected robust and frequent expression of C35 protein in breast cancer tissues. Here, we attempt to characterize the properties of C35 protein as well as to demonstrate a functional association between C35 and tumor progression. We cloned the full length gene of C35 from a breast cancer cell line, SK-BR-3, and expressed it to gain recombinant C35 protein from E. coli for circular dichroism(CD) and nuclear magnetic resonance (NMR) analyses. The CD spectrum of C35 protein revealed a possible alpha-helix secondary conformation existed. To our surprise, we also observed an extremely heat tolerance and a pH stable properties of this protein. Additionally, based on one-dimensional NMR analysis, it also showed a well organize protein structure. Finally, tertiary structure determined by two-dimensional NMR assignment reveals C35 as thioredoxin-like molecule. To warrant C35 as a cancer related gene, we first survey its expression in different cancer cell lines and tissues by using RT-PCR, Western blot and immunohistochemistry, respectively. Immunohistochemistry had showed high relevance of C35 in gastric cancer. Otherwise, survey of different cell lines by Western blotting reveals highly endogenous C35 expression in breast cancer cells, BT474. Furthermore, to investigate the function of C35 in normal tissues, analyzing of ten normal mice’s tissues reveals no detectable C35 protein. Effects of C35 on cellular proliferation, migration and apoptosis are investigating. In a preliminary test using the modified Boyden chamber assay, overexpression of C35 enables promoting cell migration toward fetal bovine serum. Beyond this, we will further establish the role of C35 in tumor progression.Abstract in Chinese…………………………………………..…………….….…..… Ibstract in English…………………………………………..…………….….…….IIIontents……………...……………………………………………………………….Vist of Figures……...…………………………………………………………..…...VIINTRODUCTION……………………………………………………………………1 1. Cancer……………………………………………………………………….1 2. Human chromosome 17q12-q21……………………………………………1 3. C35 (C17orf37) is a novel tumor biomarker………………………………..2ATERIALS and METHODS………………………….………………………......5. Antibodies…….……………………………………………..……….….......5. RNA extraction and reverser transcription-PCR………...…...……………..5. Construction of expression plasmids………………………………………..6. Preparation of purified C35 protein…………...………………………….....7. Cell lines and culture condition…………………….……………….............8. Transfection procedure…………………….………………..……………....8. Immunoprecipitation and Western blotting analyses...……………………..9. Immunohistochemistry………………………………………………….....10. Cell migration assay…………………………………………………...…..11ESULTS……………………………………………………………………………12. Construction of a novel gene, C35 (C17orf37)……………………...…….122. Structure determination of C35……………………………………………12 2.1 Circular dichroism (CD)……………………………………………...12 2.2 Nuclear magnetic resonance (NMR)…………………………………13.3 Crystallization………………………………………………………..14 3. Protein properties of C35………………………………………………….15 4. Generation of anti-C35 antibody…………………………………………..15 5. C35 protein detection by immunohistochemical staining……...………….16. C35 expression analyzed by reverse transcription (RT)–PCR…………….17. Endogenous C35 protein surveyed by Western blotting analysis……...….18. Scanning of mouse tissues for C35………………………………………..18. Cell migration assay……………………………………………….………19 ISCUSSION ……………………………………………...……………………….20IGURES……………………………………………………………………….…...24EFERENCES…………………………………………………………………..….55PPENDIX………………………………………………………………………….6