Frigola Jordi, Muñoz Mar, Clark Susan J, Moreno Victor, Capellà Gabriel, Peinado Miguel A
Molecular Oncology, IDIBELL-Institut de Recerca Oncològica, Gran Via km 2.7, Hospital Duran i Reynals, L'Hospitalet, Barcelona 08907, Spain.
Oncogene. 2005 Nov 10;24(49):7320-6. doi: 10.1038/sj.onc.1208883.
Inactivation of specific tumor suppressor genes by transcriptional silencing associated with hypermethylation of the promoter is a common event in cancer. We have applied the amplification of intermethylated sites (AIMS) technique to a 100 human colorectal cancers and seven cell lines to identify recurrent alterations that may unveil silenced tumor suppressor genes. Bisulfite sequencing was used to confirm differential DNA methylation results. Gene expression analysis was performed by real-time RT-PCR. An AIMS band recurrently displayed in tumors but not in normal tissues was isolated and identified as part of the CpG island of the prostacyclin synthase (PTGIS) gene promoter. PTGIS promoter was hypermethylated in 43 out of 100 colorectal cancers and in all cell lines. Bisulfite sequencing and clonal analysis confirmed the results obtained by AIMS and demonstrated biallelic hypermethylation of PTGIS promoter. Hypermethylation of the PTGIS promoter was associated with diminished gene expression, that was restored after treatment with demethylating and histone deacetylases inhibitor agents. PTGIS hypermethylation was associated with aneuploidy and p53 mutations. In the adjusted model, PTGIS methylation, but not p53 mutation, maintained the association with aneuploidy. We conclude that epigenetic inactivation of the PTGIS gene is a recurrent alteration in colorectal carcinogenesis.
与启动子高甲基化相关的转录沉默导致特定肿瘤抑制基因失活，这在癌症中是常见现象。我们将甲基化位点扩增（AIMS）技术应用于100例人类结直肠癌和7种细胞系，以识别可能揭示沉默肿瘤抑制基因的反复改变。亚硫酸氢盐测序用于确认DNA甲基化差异结果。通过实时RT-PCR进行基因表达分析。分离出一条在肿瘤中反复出现但在正常组织中未出现的AIMS条带，并鉴定为前列环素合酶（PTGIS）基因启动子CpG岛的一部分。100例结直肠癌中有43例以及所有细胞系中PTGIS启动子均发生高甲基化。亚硫酸氢盐测序和克隆分析证实了AIMS获得的结果，并证明PTGIS启动子存在双等位基因高甲基化。PTGIS启动子高甲基化与基因表达减少相关，用去甲基化和组蛋白脱乙酰酶抑制剂处理后基因表达得以恢复。PTGIS高甲基化与非整倍体和p53突变相关。在调整模型中,PTGIS甲基化而非p53突变维持了与非整倍体的关联。我们得出结论，PTGIS基因的表观遗传失活是结直肠癌发生过程中的一种反复改变。
