Quantitation of site-specific HPV 16 DNA methylation by pyrosequencing
Quantitation of site-specific HPV 16 DNA methylation by pyrosequencing
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DOI：
10.1016/j.jviromet.2006.08.012
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发表时间：
2006-12-01
影响因子：
3.1
通讯作者：
Unger, Elizabeth R.
中科院分区：
文献类型：
--
作者：
Rajeevan, Mangalathu S.;Swan, David C.;Unger, Elizabeth R.
人乳头瘤病毒（HPV）是宫颈癌的必要但不充分的原因。影响转录的因素，如通过病毒DNA甲基化导致的表观遗传沉默，可能影响肿瘤的进展。利用不同转录活性的细胞系CaSki（类似于400个HPV 16的整合拷贝）和SiHa（1-2个HPV 16的整合拷贝），应用焦磷酸测序技术量化HPV 16 DNA L1 3'和长控制区（LCR）上19个胞嘧啶鸟嘌呤二核苷酸（CpG）位点的甲基化。在整个19个CpG位点上，CaSki的甲基化水平从20%到100%不等，SiHa的甲基化水平从0%到85%不等，它们的启动子和增强子区域的甲基化水平差异高达40倍（SiHa < 2%, CaSki < 79%）。该方法在1-4个HPV 16 DNA拷贝/3000个细胞的极限稀释下是成功的，这一水平与大多数临床样品兼容。结果不受甲醇基细胞学收集液固定或提取方法的影响。用细胞系优化的条件适用于固定脱落宫颈细胞。焦磷酸测序可在不需要克隆的情况下对多个CpG位点的甲基化进行定量的位点特异性评估，因此适合于大规模的分子流行病学研究。Elsevier B.V.出版
Human papillomavirus (HPV) is a necessary but insufficient cause of cervical cancer. Factors influencing transcription, such as epigenetic silencing through viral DNA methylation, may impact neoplastic progression. Pyrosequencing technology was applied to quantify methylation at 19 cytosine guanine dinucleotide (CpG) sites in the L1 3' and long control region (LCR) of HPV 16 DNA using cell lines, CaSki (similar to 400 integrated copies of HPV 16) and SiHa (1-2 integrated copies of HPV 16) that differ in their transcriptional activity. Methylation levels ranged from 20 to 100% in CaSki and from 0 to 85% in SiHa over the entire 19 CpG sites, with a > 40-fold difference in the methylation levels of their promoter and enhancer regions (SiHa < 2% and CaSki 79%). The method was successful at a limiting dilution of 1-4 HPV 16 DNA copies/3000 cells, a level compatible with most clinical samples. The results were not affected by fixation in methanol-based liquid cytology collection fluid or method of extraction. Conditions optimized with cell lines were applicable to fixed exfoliated cervical cells. Pyrosequencing provides a quantitative site-specific assessment of methylation at multiple CpG sites without cloning, and is thus suited to large-scale molecular epidemiologic studies. Published by Elsevier B.V.