Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression.

启动子甲基化谱已被提议作为宫颈癌潜在的预后及/或诊断生物标志物。截至目前，学界对肿瘤发生过程中干细胞（Stem Cell, SC）标志物的启动子甲基化谱与表达模式仍知之甚少。本研究旨在明确宫颈癌变过程中干细胞基因的甲基化谱特征。全基因组启动子甲基化筛查显示，与正常外宫颈组织相比，宫颈癌组织中未分化细胞转录因子1（Undifferentiated cell Transcription Factor 1, UTF1）的启动子存在显著高甲基化。通过对液基细胞学样本提取的DNA进行直接亚硫酸氢盐焦磷酸测序，我们发现UTF1启动子甲基化水平随病变严重程度逐渐升高，在癌组织中达到峰值。该高甲基化现象与UTF1的mRNA及蛋白表达上调显著相关。通过定量逆转录聚合酶链反应（Quantitative RT-PCR）与蛋白质免疫印迹（Western Blot）实验，我们证实上皮性癌细胞系中均存在UTF1的mRNA与蛋白表达，即便其两种已被报道的主要调控因子八聚体结合转录因子4A（Oct4A）与性别决定区Y框蛋白2（Sox2）缺失时亦是如此。此外，通过免疫荧光实验，我们确认UTF1在细胞系中定位于细胞核内。令人意外的是，直接亚硫酸氢盐焦磷酸测序结果显示，在4株受试宫颈癌细胞系中，有2株经5-氮杂-2'-脱氧胞苷抑制DNA甲基转移酶后，UTF1基因的甲基化水平与表达量均出现下调。上述研究结果强烈提示，UTF1启动子甲基化谱可作为宫颈癌诊断的有效生物标志物，同时也提出了两个值得深入探讨的科学问题：一是UTF1在上皮癌变过程中发挥的具体作用，二是调控其表达的分子机制。