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研究生:傅綜信
研究生(外文):Tsung-Shin Fu
論文名稱:肺腺癌低量表現基因MOSC2(MolybdenumcofactorsulfuraseC-terminalcontaining2)之功能性探討
論文名稱(外文):Functional analysis of a lung adenocarcinoma down-regulated gene, MOSC2
指導教授:賴金美
指導教授(外文):Jin-Mei Lai
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:83
中文關鍵詞:肺癌低量表現基因MOSC2功能性探討
外文關鍵詞:Lung adenocarcinomadown-regulated geneMOSC2Functional analysis
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  • 被引用被引用:0
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中文摘要
肺癌為台灣女性頭號殺手。根據統計,這些罹患肺腺癌的女性高達85%與抽菸無關,因此推斷肺腺癌的耗發可能與自身的基因體特異有關。利用基因微陣列(Microarray)大規模分析18個女性肺腺癌病人的肺癌組織與鄰近正常組織的基因體,經統計分析後我們發現MOSC2為一個在腫瘤(tumor part)相較於鄰近正常組織(adjacent normal part)具顯著差異的基因(p value<0.0001),在定量反轉錄聚合酶連鎖反應(Q-RT-PCR)也進一步確定了此結果(p value=0.001)。由於MOSC2為一未知功能的基因,且相較於鄰近正常組織,其在腫瘤組織的表現量為低,又座落於染色體容易發生突變或缺失(deletion)的區域上,即染色體的LOH(loss of heterozygousity),故推測其功能可能與肺腺癌的產生有關。為了解MOSC2基因是否因突變或缺失致使其基因產物無法表現正常功能,因此針對13個肺癌細胞株與25個病人基因體做MOSC2基因7個表現子(exon)的序列分析,本論文共發現6個點突變(T297T/C; T360T/C, T360T; G528A /G, G528A; G730G/A, G730G; C827C/T and G933G/A),其中有5個經比對NCBI SNP database後確定為SNP(single nucleotide polymorphysm),只有一個為新找出之突變(C827C/T),但由於此突變除了細胞株H460,於其他細胞株或肺癌組織並沒有發現,因此我們認為在肺腺癌低表現量的MOSC2基因並非突變所致。另外我也建立了酵母菌雙合雜交系統期藉此找出與MOSC2發生交互作用的蛋白質以推測其功能。APOAI為本論文找出會與MOSC2胺基端結合之蛋白質。利用免疫螢光染色觀察後發現,MOSC2蛋白質表現於粒線體。為評估MOSC2蛋白質是否影響細胞週期,我們觀察過量表現於H1299細胞株之MOSC2蛋白質,似乎並不影響細胞週期各時期的分布。最後,我也嘗試找尋MOSC2基因的啟動子(promoter)區域,以了解其轉錄之調控,我們構築4個分別從轉錄起始點(Transcription start site)上游-1到-512,-1到-1003,-1到-1512以及-1到-2000的四個pGL3 basic的質體,轉染至細胞,收取細胞進行啟動子活性分析後,推論可能存在有TATA box的-512到-1003之間為MOSC2基因之啟動子區域。
Lung adenocarcinoma is the leading cause of female cancer death in Taiwan. It was noticed that 85 percent of them are nonsmokers, indicating the higher risk of these females may as least, in part, impute to their distinctive genome. Recently, 18 pairs of lung adenocarcinoma tissues and their adjacent normal tissues were analyzed by Affmatrix microarray. After data mining, we have found that MOSC2 is one of the genes whose mRNA expression is lower in tumor as compared to adjacent noncancerous part (p value<0.0001), which is also confirmed by quantitative RT-PCR(p value=0.0001). MOSC2 is a novel gene. As MOSC2 is down-regulated in lung cancer tissues, and locates on LOH(loss of heterozygousity) region, we therefore investigate the function of MOSC2 by which may shed some light on the research of lung adenocarcinoma. In attempt to knowing whether MOSC2 gene was mutated in tumor tissue, the coding sequence of MOSC2 in genome of 13 lung cancer cell lines and 25 lung cancer patients were sequenced. In this thesis, six mutations (T297T/C; T360T/C, T360T; G528A /G, G528A; G730G/A, G730G; C827C/T and G933G/A) sites have been identified. However, five out of six mutations are demonstrated as SNP in NCBI SNPs database, only one site, C827C/T, is the new mutation found in lung cancer cell line H460. We conclude that mutation is not the cause which down-regulate MOSC2 gene in lung adenocarcinoma. To investigate the function of MOSC2, I have established a yeast two-hybrid system to identify its interacting protein(s). APOAI was identified as the interacting protein of MOSC2 interact with the N-terminal region of MOSC2. Subsequently, I also performed immunofluorescence to find the subcellular localization of MOSC2. Interestingly, MOSC2 seemed to locate in mitochondria, by which is colocalized with pEYFP-mito. To evaluate the role of MOSC2 in cell cycle regulation, we also over-expressed MOSC2 in H1299 cells. However, no obvious role was found in cell cycle distribution. Finally, I try to identify the promoter region of MOSC2 toward understanding the transcriptional control of it. From the transcription start site of MOSC2, we have constructed -1 to -512, -1 to -1003, -1 to -1512 and -1 to -2000 region from genomic sequence and analyzed their promoter activity. We have concluded that -512 to -1003 which existed putative TATA box and displayed higher promoter activity may be the promoter region of MOSC2.
目錄
中文摘要………………………………………………….….......….I
Abstract………………….……………………….…………….... II
縮寫表…………………………………………………………………. III
壹、序論………………………………………….….…… ……………1
貳、實驗材料…………………………………………………………….11
參、實驗方法…………………………………………………………….16
肆、結果………………………………………………………………….29
伍、討論………………………………………………………………….37
陸、圖表……………………………………………………………….…42
柒、附錄……………………………………………………….…………69
捌、參考文獻…………………………………………………………….77
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