Department of Obstetrics and Gynecology, Peking University People's Hospital, Beijing 100044, China.
Gynecol Oncol. 2012 Mar;124(3):534-41. doi: 10.1016/j.ygyno.2011.11.023. Epub 2011 Nov 21.
The human papillomavirus (HPV) oncoprotein, E6, activates telomerase reverse transcriptase (TERT) expression and causes cellular immortalization. It remains unclear whether E6 affects TERT transcription by altering DNA methylation profiles. In this study, we explored the methylation status of the TERT promoter in cervical cancer cell lines and its variations after E6 was silenced by RNAi.
Three kinds of cervical cell lines (HPV16 positive: CaSki and SiHa; HPV18 positive: HeLa), were taken to analyze the methylation status of the TERT promoter by methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing (BS). Stealth RNAi was transiently transfected to these cell lines to silence the expression of HPV16/18 E6, and the subsequent changes of TERT mRNA levels and TERT promoter DNA methylation were examined.
Hypomethylation of the DNA around the TERT transcription start site (-156 to +162 bp) was functionally related to its transcription. After transfection with Stealth RNAi, the levels of HPV16/18 E6 and TERT mRNA were greatly decreased. The methylated CpG around the transcription start sites in CaSki and SiHa cells were statistically increased (respectively P=0.016, P=0.000). However, there was no significant difference in HeLa cells (P=0.128).
Hypomethylated CpG around the transcription start site enables the expression of TERT in cervical cancer cells. Our results show for the first time that HPV16 E6 can promote TERT transcription through demethylating the DNA sequence around the TERT transcription start site in cervical squamous cancer cells.
人乳头瘤病毒（HPV）致癌蛋白 E6 激活端粒酶逆转录酶（TERT）的表达，导致细胞永生化。目前尚不清楚 E6 是否通过改变 DNA 甲基化谱来影响 TERT 转录。本研究探讨了宫颈癌细胞系中端粒酶逆转录酶启动子的甲基化状态及其在 E6 通过 RNAi 沉默后发生的变化。
采用甲基化特异性聚合酶链反应（MSP）和亚硫酸氢盐测序（BS）分析三种宫颈细胞系（HPV16 阳性：CaSki 和 SiHa；HPV18 阳性：HeLa）中端粒酶逆转录酶启动子的甲基化状态。瞬时转染 Stealth RNAi 以沉默 HPV16/18 E6 的表达，随后检测 TERT mRNA 水平和 TERT 启动子 DNA 甲基化的变化。
TERT 转录起始位点（-156 至+162bp）周围的 DNA 低甲基化与其转录功能相关。转染 Stealth RNAi 后，HPV16/18 E6 和 TERT mRNA 的水平显著降低。CaSki 和 SiHa 细胞中起始转录位点周围的甲基化 CpG 明显增加（分别为 P=0.016，P=0.000）。然而，HeLa 细胞中没有显著差异（P=0.128）。
起始转录位点周围的低甲基化 CpG 使 TERT 在宫颈癌细胞中表达。我们的结果首次表明，HPV16 E6 可以通过去甲基化 TERT 转录起始位点周围的 DNA 序列来促进宫颈鳞状癌细胞中 TERT 的转录。
