Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan.
Biochem Biophys Res Commun. 2011 Jul 22;411(1):162-7. doi: 10.1016/j.bbrc.2011.06.121. Epub 2011 Jun 23.
Identification and characterization of epigenetically silenced genes is important for cancer research, because information from hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses and genes frequently methylated in a tumor-specific manner can be used as tumor markers. Here, we describe the identification of transcriptionally silenced hypermethylated genes in pancreatic cancer cells by using a novel method called "microarray coupled with methyl-CpG targeted transcriptional activation" (MeTA-array for short), which can effectively reactivate genes containing the stringent criteria of CpG islands at promoter regions. Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2 and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE as a control, were examined with this method, and 19 genes were upregulated twofold or more in all the three cancer cell lines after MeTA; 16 of these 19 genes have not been detected previously when using a conventional DNA demethylating agent, 5-aza-2'-deoxycytidine. Among these 16 genes, CSMD2, SLC32A1, TMEM204 and TRH were further analyzed by methylation-specific PCR, and we found that 90% (19/21) of CSMD2, 100% (21/21) of SLC32A1, 95% (20/21) of TMEM204 and 100% (21/21) of TRH were methylated in our series of pancreatic cancer cell lines. Furthermore, CSMD2, SLC32A1 and TRH were also hypermethylated in primary pancreatic cancers in a tumor-specific manner. These results suggest that MeTA-array is a highly efficient method for identifying methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer.
鉴定和描述基因的表观遗传沉默对于癌症研究非常重要，因为来自超甲基化基因的信息为理解表观遗传学在肿瘤发生中的作用提供了线索，并且在肿瘤中以特定方式频繁甲基化的基因可以用作肿瘤标志物。在这里，我们描述了一种称为“微阵列与甲基化 CpG 靶向转录激活相结合”（简称 MeTA-array）的新方法，通过该方法鉴定胰腺癌细胞中转录沉默的超甲基化基因。该方法可以有效地重新激活含有启动子区域严格 CpG 岛标准的基因。使用该方法检查了三个代表性的胰腺癌细胞系，AsPC-1、MIA PaCa-2 和 PANC-1，以及作为对照的正常胰腺导管上皮细胞系 HPDE。在 MeTA 之后，所有三种癌细胞系中均有 19 个基因的表达上调了两倍或更多；在使用常规 DNA 去甲基化剂 5-aza-2'-脱氧胞苷时，这些基因中有 16 个以前没有检测到。在这 16 个基因中，CSMD2、SLC32A1、TMEM204 和 TRH 进一步通过甲基化特异性 PCR 进行分析，我们发现 90%（19/21）的 CSMD2、100%（21/21）的 SLC32A1、95%（20/21）的 TMEM204 和 100%（21/21）的 TRH 在我们的一系列胰腺癌细胞系中被甲基化。此外，CSMD2、SLC32A1 和 TRH 在肿瘤特异性的原发性胰腺癌中也发生了超甲基化。这些结果表明，MeTA-array 是一种高效的方法，可用于鉴定人类胰腺癌中甲基化介导的转录沉默基因，并且该方法可应用于其他类型的人类癌症。
