Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, PR China.
Oncol Rep. 2012 Jul;28(1):99-104. doi: 10.3892/or.2012.1779. Epub 2012 Apr 23.
To identify novel hypermethylated genes in colorectal cancer (CRC) and to test their potential application in CRC early diagnosis, a genome-wide screening of 57,723 CpG dinucleotides covering 4,010 genes was performed using MIRA-based microarrays in paired DNA samples extracted from 3 fresh frozen CRC tissues and their matching non-cancer tissues from 3 CRC patients undergoing curative surgery. Candidate hypermethylated genes screened by MIRA-based microarrays were further validated in independent CRC samples. A total of 297 CpG dinucleotides covering 211 genes were found to be hypermethylated in CRC tissues. From these 211 candidate methylated genes, three novel hypermethylated genes with more than four probes positive were picked up for validation. Direct bisulfite sequencing revealed that methylations occurred at multiple CpG sites of these three genes in cancer tissues, especially for PHOX2B and FGF12. Combined bisulfite restriction analysis showed that these three genes were methylated in cancer samples but not in non-cancer samples. We also compared the methylation levels of these three novel hypermethylated genes with those of vimentin and SEPT9, well-known hypermethylated genes in CRC, and found that methylated PHOX2B, FGF12 and GAD2 were better than methylated vimentin and SEPT9 in differentiating CRC cancer tissue from non-cancer tissue. Significant enrichment analysis of GO terms of the hypermethylated genes showed that a high proportion of hypermethylated genes in cancer tissues are involved in the regulation of transcription. In conclusion, we found a set of novel hypermethylated genes in CRC, which may have potential to be used as biomarkers for the early diagnosis of CRC.
为了鉴定结直肠癌（CRC）中新型的高甲基化基因，并检验它们在 CRC 早期诊断中的潜在应用，我们采用基于 MIRA 的微阵列对来自 3 位接受根治性手术的 CRC 患者的 3 对新鲜冷冻 CRC 组织及其匹配的非癌组织的 DNA 样本进行了 57723 个 CpG 二核苷酸的全基因组筛查，该微阵列共覆盖了 4010 个基因。通过 MIRA 微阵列筛选的候选高甲基化基因在独立的 CRC 样本中进一步得到验证。在 CRC 组织中发现了 297 个 CpG 二核苷酸覆盖了 211 个基因发生了高甲基化。在这 211 个候选甲基化基因中，挑选了 3 个有超过 4 个探针呈阳性的新的高甲基化基因进行验证。直接亚硫酸氢盐测序显示，这些基因在癌症组织中的多个 CpG 位点发生甲基化，尤其是 PHOX2B 和 FGF12。联合亚硫酸氢盐限制分析显示，这些基因在癌症样本中发生甲基化，但在非癌样本中未发生甲基化。我们还将这三个新的高甲基化基因的甲基化水平与 CRC 中已知的高甲基化基因 vimentin 和 SEPT9 进行了比较，发现甲基化的 PHOX2B、FGF12 和 GAD2 比甲基化的 vimentin 和 SEPT9 更能区分 CRC 癌组织和非癌组织。对高甲基化基因的 GO 术语进行显著富集分析显示，癌症组织中高甲基化基因的很大一部分参与转录调控。总之，我们在 CRC 中发现了一组新的高甲基化基因，它们可能具有作为 CRC 早期诊断标志物的潜力。
