Genome-wide analysis in human colorectal cancer cells reveals ischemia-mediated expression of motility genes via DNA hypomethylation (expression). Homo sapiens

DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumors, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel results suggest that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes. Overall design: HCT116 cells were grown in either Hypoxic or Hypoglycaemic conditions, and compared to cells grown under normal conditions

DNA低甲基化（DNA hypomethylation）是一类重要的表观遗传修饰，已在多种癌症类型中被检出，可引发既往沉默基因的上调表达以及基因组稳定性的丧失。本团队前期研究证实，实体瘤微环境中常见的两种变化——缺氧与低糖（缺血），可通过下调人类结肠癌细胞内的DNA甲基转移酶（DNMTs）降低DNA甲基化水平。本研究采用全基因组跨平台策略，旨在鉴定缺血诱导的低甲基化且表达上调的基因。将人类结肠癌细胞（HCT116）暴露于缺氧或低糖环境后，对其甲基化DNA进行免疫沉淀，并通过Affymetrix启动子芯片开展分析；与此同时，分离细胞总RNA并通过Affymetrix表达芯片进行平行检测。通过Ingenuity通路分析（Ingenuity pathway analysis）软件分析发现，大量低甲基化且表达上调的基因参与细胞运动过程，其中包括PLAUR与CYR61。基质胶（Matrigel）侵袭实验证实，在缺氧或低糖条件下培养的HCT116细胞迁移能力显著增强。通过实时定量聚合酶链式反应（qPCR）验证了细胞运动相关基因的表达上调情况。缺血与癌症转移的相关性在癌症进展领域已得到广泛证实，但该现象背后的分子机制尚未被明确阐明。本研究的创新性结果表明，缺氧与低糖可能通过下调DNMTs驱动DNA甲基化状态的改变。据我们所知，本研究首次为缺血细胞的转移潜能增强提供了合理解释：即缺血可诱导DNA低甲基化，并上调细胞运动相关基因的表达。实验整体设计：将HCT116细胞分别置于缺氧、低糖条件下培养，并与正常培养条件下的细胞进行对照分析。