以台灣肺癌患者胸水中建立出之HPV16感染之肺腺癌細胞株探討HPV16E6在肺癌p16基因甲基化作用機轉

中山醫學大學機構典藏 CSMUIR > 醫學院 > 醫學系 > 研究計劃 > Item 310902500/2854

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题名:	以台灣肺癌患者胸水中建立出之HPV16感染之肺腺癌細胞株探討HPV16E6在肺癌p16基因甲基化作用機轉 Study of Hpv 16 E6 in P16 Gene Promoter Hypermethylation in Tainewase Lung Cancer Cell Culture from Pleuraleffusion
作者:	吳銘芳;李輝;鄭雅文 Wu, Ming-Fang;Cheng, Yawen
贡献者:	中山醫學院醫學系
关键词:	肺癌;p16基因 Lung cancer: p16;HPV16E6;DNMT3b
日期:	2008
上传时间:	2010-11-25T03:11:22Z (UTC)
摘要:	最近研究指出，致癌病毒感染宿主細胞，會經由使宿主細胞抑癌基因甲基化，而導致細胞的腫瘤化，但其作用機轉仍不清楚。已知高危險型HPV 所轉錄產生的致癌蛋白E6 可經由降解p53 抑癌蛋白而促使細胞的腫瘤化，亦有研究指出p53 抑癌蛋白可抑制DNMT 轉錄起始區的轉錄活性進而抑制DNMT 蛋白的表現，因此推測 HPV 可能藉由E6 蛋白使p53 蛋白去活化而無法抑制DNMT 的轉錄，進而促使抑癌基因p16 的甲基化。但在子宮頸癌的研究發現，HPV 16/18 E7 會與HDAC 交互作用，而使HDAC 維持DNA 雙股結構之能力失效，使E2F 啟動有關細胞增生之基因轉錄活化。這顯示HPV 16/18 E6 及E7 致癌蛋白在肺癌細胞及子宮頸癌細胞株的甲基化調控機制以及致癌機轉有所不同。除 p53 外，DNMT3b 轉錄起始區的基因多型性亦影響其基因的轉錄調控，且HPV16 致癌蛋白E6 是否會直接調控DNMT3b 的表現則仍不清楚，因此本計畫擬構築不同基因型之DNMT3b 轉錄起始區轉染至肺腺癌細胞株A549 ，利用report assay 及 Chromatin immunopreciption assay (CHIP) 方式分析釐清HPV E6 致癌蛋白是經由抑制p53 而活化DNMT3b，或是可直接經由與DNMT3b 轉錄起始區結合而活化 DNMT 的表現，並釐清HPV 對不同基因型之DNMT3b 的轉錄活性是否有影響，此外亦利用MS-PCR 及同步定量反轉錄聚合酶連鎖反應(real-time RT-PCR)分別偵測 p16基因的甲基化及其mRNA 表現，以證明HPV 確實經由影響DNMT3b 造成p16 基因甲基化影響p16 抑癌基因表現而使細胞腫瘤化。 Frequent promoter hypermthylation of tumor suppressor genes (TSGs), such as p16, RASSF1A, and ER, was frequently observed in oncogenic virus-induced human cancers. However, the underline mechanism is still unclear. TSGs transcription was regulated by the balance of acetylation and deactylation of histone through histone acetylase (HAT) and histone deacetylase (HDAC), respectively. In cervical cancer, HPV 16/18 E7 can be interacted with HDAC to open DNA strand for E2F binding and then upregulates cellproliferation-associated gene expressions. Our preliminary data showed that HDAC expression was increased in lung cancer cells after transfected with HPV 16 E6, E7, respectively, and the hypermethylation frequency of p16 and RASSF1A was concomitantly increased in HPV-transfected lung cancer cells as compared with parental cells. Moreover, the expression levels of DNMT1 and DNMT3b in cervical cancers were significantly lower than those of lung tumors with HPV infection (unpublished data). This result was consistent with fewer p16 hypermethylation in cervical cancer compared to lung cancer. Based on the above mentions, HPV infections in lung cancer and cervical cancer had a different role in p16 inactivation. Beside p53，DNMT3b promoter polymorphism will also affect on p16 transcriptional regulation, and whether HPV16 E6 oncoprotein can regulate DNMT3b expression directly or not is still unclear. Therefore, we propose to construct different genomic types of DNMT3b promoters and transfect them into lung adenocarcinoma cells A549, by using report assay and Chromatin immunopreciption assay (CHIP) to illuminate whether HPV E6 oncoprotein is through the inhibition of p53 and further activation of DNMT3b, or through binding with DNMT3b promoter directly to activate DNMT expression, also to clarify whether HPV will affect the regulatory activity of different genomic types of DNMT3b promoters or not. Meanwhile, p16 hypermethylation and its mRNA expression will be examined by MS-PCR and real-time RT-PCR, respectively, to prove that HPV is through DNMT3b upregulation and then causes p16 hypermethylation and tumorigenesis.
URI:	https://ir.csmu.edu.tw:8080/handle/310902500/2854
显示于类别:	[醫學系] 研究計劃

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