Hypomethylated CpG around the transcription start site enables TERT expression and HPV16 E6 regulates TERT methylation in cervical cancer cells

Objective. 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.Methods. 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.Results. 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).Conclusion. 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. (C) 2011 Elsevier Inc. All rights reserved.

目的。人乳头瘤病毒（HPV）癌蛋白E6激活端粒酶逆转录酶（TERT）的表达并导致细胞永生化。目前尚不清楚E6是否通过改变DNA甲基化谱来影响TERT转录。在本研究中，我们探讨了宫颈癌细胞系中TERT启动子的甲基化状态以及在通过RNA干扰（RNAi）沉默E6后其变化情况。 方法。选取三种宫颈细胞系（HPV16阳性：CaSki和SiHa；HPV18阳性：HeLa），通过甲基化特异性聚合酶链反应（MSP）和亚硫酸氢盐测序（BS）分析TERT启动子的甲基化状态。将 stealth RNAi瞬时转染到这些细胞系中以沉默HPV16/18 E6的表达，并检测TERT mRNA水平和TERT启动子DNA甲基化的后续变化。 结果。TERT转录起始位点周围（-156至 + 162 bp）的DNA低甲基化与其转录功能相关。用stealth RNAi转染后，HPV16/18 E6和TERT mRNA的水平大幅下降。CaSki和SiHa细胞转录起始位点周围的甲基化CpG在统计学上增加（分别为P = 0.016，P = 0.000）。然而，在HeLa细胞中没有显著差异（P = 0.128）。 结论。转录起始位点周围的低甲基化CpG使TERT在宫颈癌细胞中得以表达。我们的结果首次表明，HPV16 E6可通过使宫颈鳞状癌细胞中TERT转录起始位点周围的DNA序列去甲基化来促进TERT转录。（C）2011爱思唯尔公司。保留所有权利。