Methylation analysis of multiploid colorectal cancer for the epigenetic therapy establishment

DNA多倍体肿瘤是指肿瘤细胞群中二倍体和非整倍体在同一区域内共存，是一种有用的肿瘤进展分析模型。在本研究中，我们使用多倍体结肠直肠癌，试图阐明p53和Ki-ras突变，以及癌症相关基因甲基化与二倍体到非整倍体进展之间的关系。采用隐窝分离技术和流式细胞术细胞分选，检测了33例多倍体结直肠癌，并将多倍体群体分为无间质组织的二倍体和非整倍体群体。在每个人群中，我们通过聚合酶链反应法提取和扩增DNA，然后分析Ki-ras和p53突变以及癌症相关基因甲基化状态。采用单链构象多态性和直接测序进行P53突变分析。Ki -ras突变进行直接测序分析。癌症相关基因启动子区域的甲基化分析采用亚硫酸氢盐限制性结合法进行。非整倍体群体中p53基因突变发生率为63.2%，高于二倍体群体（1.0.5%）。非整倍体群体中ZFP64、HPP1和RASSF2A的甲基化率分别为7.9%、13.2%和36.8%，高于二倍体群体。结果表明，p53突变和ZFP64、hpp1和RASSF2A的甲基化对于二倍体向非整倍体的进展是重要的。

DNA multiploid cancer is defined as the coexistence of diploid and aneuploid tumor cell populations in the same area, and is a useful model for cancer progression analysis. In this study, we used multiploid colorectal cancer and attempted to clarify p53 and Ki-ras mutations, and the correlation between cancer-related gene methylation and diploid to aneuploid progression. Using the crypt isolation technique and flow cytometric cell sorting, we detected 33 multiploid colorectal cancers, and divided multiploid populations into diploid and aneuploid populations without stromal tissue. In each population, we extracted and amplified DNA by polymerase chain reaction assay, and then analyzed Ki-ras and p53 mutations and cancer-related gene methylation status. p53 mutation analysis was performed by single-strand conformation polymorphism and direct sequencing. Ki -ras mutatio n analysis was performed by direct sequencing. Methylation analysis of cancer-related gene promoter regions was performed by combined bisulfite restriction assay . Mutations of p53 gene in aneuploid populations (63.2%) were more frequent than in diopoid populations (1 0.5%). Methylation of ZFP64 , HPP1 and RASSF2A in aneuploid populations (7.9 ％、13.2％、36.8％) was higher than in diploid populations. The results suggest that mutation of p53 and methylation of ZFP64 , HPP1and RASSF2A are important for diploid to aneuploid progression.