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研究生(外文):Bo-Min Yang
論文名稱(外文):To evaluate the correlation between methylation status of CpG island and down-regulation gene in female lung adenocarcinoma
指導教授(外文):Jin-Mei Lai
外文關鍵詞:DNA methylationCpG islandbisulfite sequencingfemale lung adenocarcinoma
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肺癌是目前台灣與世界各地癌症患者主要的死亡原因,其中肺腺癌是女性肺癌發生的最主要型態,由於85%的女性肺腺癌屬於非吸煙族群,顯示女性肺腺癌的發生可能歸咎於特殊的基因體。許多副基因體的修飾機制,例如DNA甲基化作用可能抑制抑癌基因的表現而導致癌症發生,因此我們藉由分析基因之甲基化情形,來探討其是否為女性肺腺癌中部分低量表現基因的調控機制。在18位不吸煙的女性肺腺癌患者基因微陣列(microarray)的分析中,我們鎖定八個包括FAM134B、MOSC2、PTPRM、KIAA0256、GPRC5A、CXCL5、EDNRB、PDPN在腫瘤組織中表現量顯著較鄰近正常組織為低的基因。為了要分析與呈現這些基因CpG island的甲基化狀態,我們利用酸性亞硫酸鈉(sodium bisulfite)處理基因體(genomic)DNA,將未甲基化之胞嘧啶(cytosine)轉化成尿嘧啶(uracil),利用nest PCR增幅CpG island片段後進行定序。分別分析一個正常肺細胞株IMR90與14個肺癌細胞株之上述八個基因後,我們發現MOSC2、CXCL5、EDNRB在多個肺癌細胞株呈現高度甲基化而在正常細胞株呈現低度甲基化。此外使用DNA methyltransferase抑制劑5-aza-2'-deoxycytidine處理細胞,可見隨濃度的提高,基因的表現量有逐漸回升的現象,顯示DNA甲基化作用可能確實是MOSC2、CXCL5、EDNRB在肺癌細胞株中表現量降低的調控機制。
Lung cancer is currently the leading cause of cancer death worldwide as well as in Taiwan. Adenocarcinoma is the most prevalent histological type among female lung cancer. It was noticed that 85 percent of female adenocarcinoma patients are nonsmokers, indicating their higher risks may as least, in part, impute to their distinctive genome. As epigenetic mechanism, such as DNA methylation, has been implicated in regulation of tumor suppressor genes, we therefore analyze whether DNA methylation is the mechanism that affect the gene which is down-regulated in female lung adenocarcinoma. By analyzing microarray profiling of tissue pair from 18 lung adenocarcinoma patients, eight genes including FAM134B, MOSC2, PTPRM, CXCL5, KIAA0256, GPRC5A, EDNRB, and PDPN have been found to down regulated in lung cancer tissue as compared with adjacent normal part. In attempt to analyze whether these genes have CpG islands and display hypermethylation pattern, we used sodium bisulfite conversion method to treat genomic DNA and to sequence the putative CpG island of each gene. After analyzed the methylation status of CpG islands of these genes in one normal lung fibroblast, IMR90, and fourteen lung cancer cell lines, we have found MOSC2, CXCL5 and EDNRB displayed hypermethylation in many lung cancer cell lines as compared to IMR90. In addition, DNA methyltransferase inhibitor, 5-aza-2’-deoxycitidine, can revert their down regulation in a dose dependent manner, indicating DNA methylation may indeed the mechanism that down-regulates the expression of MOSC2, CXCL5 and EDNRB in lung cancer cell lines.
中文摘要 1
Abstract 2
壹、序論 3
貳、實驗材料 15
參、實驗方法 18
肆、結果 28
伍、討論 44
陸、圖表 50
柒、附錄 73
捌、參考文獻 94
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