2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647915摘要：鼻咽癌是住在中國南方、新加坡及台灣的華人很常見的癌症之一. 至今其真正原因仍不明.環境因素,諸如多吃醃魚、多用中藥,及長期暴露於硫酸蒸氣等都被認為與此癌之發生有關. 而遺傳因素, 至今仍然沒有證據證明任何一重要基因與鼻咽癌之病理機轉有直接關聯. 此外EB 病毒的感染也被認為與鼻咽癌的發生有很密切的關係。為了要研究鼻咽癌的起癌原因, 我們實驗室在過去曾經建立過10 株鼻咽癌細胞株(其中九株已發表過),並研究其與EB 病毒的因果關係. 我們的結果顯示EB 病毒並不會直接感染鼻咽黏膜的鱗狀化生之上皮細胞. 但卻會感染癌化之細胞, 並能促進癌細胞增生. 這種結果讓我們轉向去研究鼻咽癌的基因改變. 我們利用cDNA 微矩陣核酸分析法去分析癌細胞株及正常鼻腔黏膜上皮細胞之整體基因之不同表達,因而發現了不少過高或過低表達之兩個基因群.我們收集了40 個此兩基因群之基因,利用定量反轉多聚合酶鏈反應(quantitative RT-PCR)去分析在鼻咽癌細胞及正常上皮細胞內的各個基因的表現. 其中有11 個基因被證實只有很微量的表現於鼻咽癌細胞內但很明顯的表現於正常上皮細胞. 若用較多的正常上皮細胞及更多的癌細胞株去觀察時,發現只有6 個基因有微量表現. 若用Pearson’s correlation 法去觀察此6 個基因之間是否有相互關係, 結果發現他們之間確實有相互關聯. 亦即A gene 在NPC cell 若underexpression,其他五個基因同時可見underexpression, 反之亦是. 而其互相之關聯用ANOVA 或然率的計算結果,指出其關聯性有很強的意義.當利用更多的鼻咽癌檢體之RNA 去檢測時, 發現其中只有2 個genes 都同時呈現有意義的under expression. 若用DBTSS 的database 去研究此2 個genes 之promoter 有無共同DNA binding sites 時, 發現2 個genes 皆有數個共同之motif 可受DNA binding. 之後, 我們用TRANSFAC data base 去找何種transcription factors 能與這些motif binding. 結果出現有3 個transcription factors 都能與此2 個genes 之promoter 之DNA 結合. 其中之一為SOX 5 gene.若再以此gene 去檢查NPC 之RNA 表現時, 發現此gene 皆為overexpression 的狀態. 因此我們認為此基因(SOX 5 gene)很可能同時調控其下游的2 個genes. 後者也可調控其他更下游的基因表現. 所以, 此基因很可能在NPC 的progression 中很重要, 可能是早期調控其下游基因之一. 也就是說此基因可能是鼻咽癌癌化後早期即起變化的基因. 為了深入研究此基因之生理功能, 及其如何在NPC細胞調控其他的基因, 而後者又如何調控其下游之基因, 我們也做了一些先驅研究工作. 其結果是:我們利用廠商製備的抗體發現SOX-5 只表現於NPC 細胞株. 而此基因所調控的主要下游基因為SPARC基因. FGFR 雖也受SOX-5 調控, 但不重要.由chromatin immunoprecitation 得知SOX-5 可直接與SPARC 的promoter DNA 結合而調控之, 同時也發現SOX-5 及SPARC 基因之表現及功能在NPC 上所表現的剛好相反. 即SOX-5 表現高時, SPARC 就表現低, 此時皆可促進癌細胞之增生. 當我們用SOX-5的抗體對66 個NPC cases(取自1997 及1998 年的病人)的檢體用免疫組織化學染色時SOX-5 若高表現於檢體之癌細胞時, 則不分NPC 病理分類. 不論NPC 之臨床分期. 其預後存活率皆差. 但是沒有表現或表現量小時, 則不分種類,及臨床預後期別, 其5 年存活率皆不錯.從之前的實驗我們提出一假設, 高表現的SOX-5 基因可當做鼻咽癌惡化的指標. 因高表現之SOX-5 可促進NPC 細胞的增生及侵犯性, 也可能誘發鼻咽癌轉移基因的表現, 如MMP 蛋白消化酶等. 因此本計畫的研究重點將包括尋找可被SOX-5 調控的基因群及分析其功能:(1) 將INHBA (activin), VNN3 以及CCL28 等基因轉殖出來.(2) 建構包含INHBA, VNN3 以及CCL28 等基因於可誘導表現的載體pBIG2i-SOX-5C.(3) 建立帶有pBIG2i-INHBA, pBIG2i-VNN3 以及pBIG2i-CCL28 的穩定鼻咽癌細胞株(NPC-TW04).(4) 研究INHBA, VNN3 以及CCL28 等基因的表現，對鼻咽癌細胞的生長、爬行、以及轉移等細胞生理現象之影響.(5) 將對於INHBA, VNN3 以及CCL28 等基因具有高表現力的鼻咽癌細胞株，分別注射到NODＳＣＩＤ 老鼠的皮下，觀察這些細胞株在活體中的生長及轉移的情形.(6) 觀察SOX-5, SPARC, activin, CCL28 以及VNN3 等基因於鼻咽癌、口腔癌、肝及肺癌等不同癌症的表現及其對臨床上意義.由本計畫所作研究之成果將可提供SOX-5C 基因及其調控的一群基因以供鼻咽癌在臨床上之預後指標, 同時也可用以說明早期鼻咽癌病理機轉的基本變化並可當癌症治療的標的基因.<br> Abstract: Nasopharyngeal carcinoma (NPC) is one of the most common cancers in Chinese living in South China,Singapore and Taiwan. The actual etiological factors of this cancer are still not well elucidated yet. Forhereditary factor, so far, there is no evidence supporting the hypothesis that any major gene is involvedin NPC tunorigensis. Although environmental factors, such as long-term consumption of salted fish andChinese herbs and long-term exposure to sulfuric acid vapor have been proposed to be able to induceNPC formation, other important factors are still unknown. However, Epstein-Barr virus (EBV) hasbeen proposed to be closely associated with NPC. To investigate the molecular tumorigenicity of NPC,we have established 10 NPC cell lines (9 lines have been reported), and examined the relationshipbetween EBV and NPC in the last several years. Our results indicate that although EBV can not infectand transform normal nasopharyngeal epithelial cells, but it can infect tumor cells to enhance cellularproliferation. From this conclusion, we have investigated the genetic alteration of NPC. Using cDNAmicroarray analysis to perform the global survey of differential gene expression profiling of NPC, wehave identified two sets of highly overexpressed and markedly underexpressed gene sets in NPC. Fortymarkedly upregulated and down-regulated genes are collected and subjected to quantitative-RT-PCR(Q-RT-PCR) analysis in NPC cell lines and normal epithelial cells. Eleven of them shows clearlydown-regulated in NPC cells when compared with normal epithelial cells. By Pearson’s correlation testto find if they are positively or negatively correlated with each other, six of them (FGFR1, IGFBP6,SPARC, UCHL1, RPL37A, CMTM7), reveal significantly down-regulated in NPC biopsy specimens.Increase of the number of NPC cell lines and the normal nasomucosal epithelial cultures, only FGFR1AND SPARC genes are significantly and correlatively down-expressed in NPC cells. Using databaseof Human Transcriptional Start Sites, some consensus DNA binding sites are identified in the promoterregions of these 2 genes. In addition, using TRANSFAC database system to identify the transcriptionfactors which can bind to the promoter regions of these 2 genes, several transcription factors includingSOX-5 gene are found to be able to bind to these 2 genes. In addition, SOX-5 gene expression is seenup regulated in most NPC cell lines and some biopsy specimens. These results suggest that SOX-5gene may be one of the major key genes in NPC tumorigenicity, which can regulate its several downstream genes and the latter may further regulate other downstream genes. These preliminary results arevery exciting and important. In 66 NPC biopsy specimens, overexpression of SOX-5 in tumor cellscorrelates clinically with poor survival.From the previous experiment we proposed that overexpressionof SOX-5 gene may be used as a prognosis factor for NPC progression, because it may promote NPCcell proliferation and invasion ability, and may induce the expression of some metastasis related gene,such as metalloproteases (MMPS), and other genes. Therefore, in the present research proposal we willperform the following experiments, including:1. Cloning of INHBA (activin), VNN3 (vanin-3) and CCL28 genes.2. Construct doxycycline (DOX) inducible expression vector pBIG2i containing INHBA, VNN3 andCCL28 genes.3. Establish stable clones of NPC-TW04 cell lines transfected with pBIG2i-INHBA, pBIG2i-VNN3and pBIG2i-CCL28 and verify these lines.4. Investigate cell proliferation rate, migration and invasion abilities in NPC-TW04 -pBIG2i-activin,NPC-TW04-pBIG2i-CCL28, and NPC-TW04-pBIG2i-VNN3 transfectants, respectively.5. In vivo investigation of NPC-TW04-pBIG2i-activin, NPC-TW04-pBIG2i-CCL28 andNPC-TW04-pBIG2i-VNN3 transfectant- transplanted xenograft growth in the NOD-SCID mice,separately.6. Observation of SOX-5, SPARC, activin, CCL28 and VNN3 expressions in different NPC cell lines,biopsy specimens and other cancer cell lines (hepatoma, lung cancer and colon cancer lines) andtheir surgical specimens, and evaluation of their clinical significance.If this experimental proposal can be accomplished, the results of this work may provide some veryvaluable information for clinical prognosis, and provide the fundamental basis for investigation of NPCearly tumorigenicity and the genetic targets for clinical intervention.