Methylomic analysis identifies epigenetically silenced genes and implies an activation of β-catenin signal in cervical cancer. Homo sapiens

Using the pools of DNA from squamous cervical carcinomas (SCC) and DNA from normal scraping cells discovered the abnormal changes of genomic wide methylation by methylation BeadChip. The 61 genes were selected to validate by quantitative methylation specific PCR (qMSP) and bisulfite pyrosequencing in further processes. A CpG islands methylator phenotype (CIMP) was confirmed at 14 candidates in an independent set contained a spectrum of scraping cells form abnormal cervical lesions. In addition, we found among of 7 genes were announced to imply to potentially biological functions of β-catenin signal. Overall design: A pool DNA collected from equal amount of DNA of 38 SCC, and the other pool collected from equal amount of DNA of 19 normal cervical scraping cells. Based on 40 percent of different methylation in two pools of samples, we selected 91 genes showed abnormal methylation in SCC. After the confirmation of gene restored expression in cervical cell lines, there 61 genes showed upregulation in the treatment with demethylating drug. In addition, gel based of MSP were contributed to confirm in a small size of samples, and quantitative MSP was contributed to validated in an independent set. The methylation status of qMSP was also confirmed by bisulfite pyrosequence. Finally, we selected 14 genes demonstrated clinical relevance in the high methylation of CIMP associated to a risk factor in cervical intraepithelial neoplasia grade 3 (CIN3) and worse lesions. isk factor in cervical intraepithelial neoplasia grade 3 (CIN3) and worse lesions.

本研究借助甲基化芯片（methylation BeadChip），对宫颈鳞状细胞癌（squamous cervical carcinomas, SCC）的DNA池与正常宫颈刮取细胞DNA进行分析，检出全基因组甲基化异常改变。后续选取61个基因，通过定量甲基化特异性PCR（quantitative methylation specific PCR, qMSP）与亚硫酸氢盐焦磷酸测序开展验证实验。在包含一系列异常宫颈病变刮取细胞的独立队列中，证实14个候选基因存在CpG岛甲基化表型（CpG islands methylator phenotype, CIMP）。此外，本研究发现其中7个基因可能与β-连环蛋白信号通路的潜在生物学功能相关。
总体实验设计：将38例宫颈鳞状细胞癌的DNA等量混合构建DNA池，另将19例正常宫颈刮取细胞的DNA等量混合构建对照DNA池。基于两个样本DNA池间40%的差异甲基化比例，筛选出91个在宫颈鳞状细胞癌中存在甲基化异常的基因。随后在宫颈细胞系中验证基因的表达恢复情况，发现经去甲基化药物处理后，其中61个基因呈现表达上调。此外，采用基于凝胶的甲基化特异性PCR对小样本队列进行验证，并通过定量甲基化特异性PCR在独立队列中进一步确认。定量甲基化特异性PCR检测的甲基化状态亦通过亚硫酸氢盐焦磷酸测序加以验证。最终筛选出14个基因，其高甲基化的CIMP表型与宫颈上皮内瘤变3级（cervical intraepithelial neoplasia grade 3, CIN3）及更严重病变的发病风险显著相关。