Methylation analysis of multiploid colorectal cancer for the epigenetic therapy establishment

DNA多倍体癌症定义为在同一区域内二倍体和非整倍体肿瘤细胞群共存，是癌症进展分析的有用模型。在这项研究中，我们使用了多倍体结直肠癌，并试图阐明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相关性。采用腺泡分离技术和流式细胞仪细胞分选技术，检测了33例大肠癌的多倍体，并将其分为二倍体和非整倍体两组。在每个人群中，我们提取并扩增DNA聚合酶链反应，然后分析Ki-ras和p53突变和癌症相关基因甲基化状态。通过单链构象多态性和直接测序进行p53突变分析。直接测序法检测Ki -ras基因突变.采用亚硫酸氢盐限制性酶切分析法对癌相关基因启动子区进行甲基化分析。非整倍体人群p53基因突变率（63.2%）高于二倍体人群（10.5%）。ZFP 64、HPP 1和RASSF 2A基因甲基化水平在非整倍体群体中分别为7.9%、13.2%和36.8%，高于二倍体群体。结果提示，p53基因突变和ZFP 64、HPP 1和RASSF 2A基因甲基化在二倍体向非整倍体转化中起重要作用。

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.