Human Telomerase Reverse Transcriptase Activated by E6 Oncoprotein Is Required for Human Papillomavirus-16/18-Infected Lung Tumorigenesis
Human Telomerase Reverse Transcriptase Activated by E6 Oncoprotein Is Required for Human Papillomavirus-16/18-Infected Lung Tumorigenesis
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DOI：
10.1158/1078-0432.ccr-08-0850
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发表时间：
2008-11-15
影响因子：
11.5
通讯作者：
Lee, Huei
中科院分区：
文献类型：
--
作者：
Cheng, Ya-Wen;Wu, Tzu-Chin;Lee, Huei
目的：我们最近的研究表明，人乳头瘤病毒（HPV）-16/18 E6癌蛋白在肺肿瘤中表达，并与p53失活有关。实验设计：免疫组化检测135例肺肿瘤组织中HPV-16 E6蛋白表达，实时荧光定量PCR检测hTERTmRNA表达，原位杂交检测hTERTmRNA表达。小RNA干扰（RNAi），蛋白质印迹和染色质免疫沉淀分析被用来澄清hTERT转录是否由c-Myc和Sp1调节。采用实时荧光定量端粒重复序列扩增法和软琼脂法分别检测E6或hTERT-RNAi对TL-1的端粒酶活性和致瘤潜力。结果：在女性、非吸烟者和腺癌中，EIS阳性肿瘤中hTERTmRNA水平显著高于E6阴性肿瘤。此外，早期肿瘤（1期）中的hTERT mRNA水平高于晚期肿瘤（11期和111期）中的水平。染色质免疫沉淀实验表明，Sp1与c-Myc协同作用激活TL-1细胞中hTERT的转录，与SiHa细胞相似。转染不同剂量的E6-或hTERT-RNAi后，TL-1细胞的端粒酶活性下降。软琼脂实验表明，转染E6-RNAi后的TL-1细胞的致瘤能力显著降低。此外，用hTERT-RNAi转染后的TL-1细胞不能形成集落。结论：HPV-16/18感染的肺肿瘤发生需要E6癌蛋白转录激活hTERT。
Purpose: Our recent report indicates that human papillomavirus (HPV)-16/18 E6 oncoprotein is expressed in lung tumors and is related to p53 inactivation. We further explored whether human telomerase reverse transcriptase (hTERT) transcription is up-regulated by E6 and contributes to lung tumor development.Experimental Design: Immunohistochemistry detected HPV-16 E6 oncoprotein in 135 lung tumors, and hTERT mRNA was evaluated by real-time reverse transcription-PCR and in situ hybridization, respectively. A small RNA interference (RNAi), Western blotting, and chromatin immunoprecipitation analysis were used to clarify whether hTERT transcription was regulated by c-Myc and Sp1. The telomerase activity and oncogenic potential of TL-1 with or without E6- or hTERT-RNAi was determined by real-time quantitative telomeric repeat amplification protocol analysis and soft-agar assay, respectively.Results: hTERT mRNA levels in EIS-positive tumors, which were prevalent in females, nonsmokers, and adenocarcinomas, were significantly higher than in E6-negative tumors. In addition, hTERT mRNA levels in early tumors (stage 1) were greater than levels in advanced tumors (stages 11 and 111). Chromatin immunoprecipitation assay showed that Sp1 cooperated with c-Myc to activate hTERT transcription in TL-1 cells, which was similar to the SiHa cells. The telomerase activity of the TL-1 cells decreased concomitantly with the transfection of various doses of E6- or hTERT-RNAi. A soft-agar assay showed that the oncogenic potential of TL-1 cells was significantly reduced after being transfected with E6-RNAi. Moreover, a colony of TL-1 cells could not form after transfection with hTERT-RNAi. Conclusion: Transcriptional activation of hTERT by E6 oncoprotein is required for HPV-16/18-infected lung tumorigenesis.