Identification of genes upregulated by MeTA-array in human pancreatic cancers

Identification and characterization of epigenetically silenced genes is very important for cancer research. Particularly, information of 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. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2’-deoxycytidine were widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting such methylation-mediated silenced genes in cancer and successfully developed a new method termed “methyl-CpG targeted transcriptional activation (MeTA)” by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD) that specifically recognizes and binds to methylated DNAs. Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the present study, we applied this method to three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control). All of these cell lines have already been analyzed their expression profiles by 5-aza-2’-deoxycytidine. We first analyzed the expression of five genes by RT-PCR with Southern hybridization, NEFH, NPTX2, SFRP1, TIMP3, and UCHL1; these genes are known to be methylated in at least any one of these cancer cell lines. Upregulation by “MeTA” was confirmed in all of these genes. Then we searched for upregulated-genes, by two-folds or more, in all the three cancer cell lines after MeTA; nineteen such upregulated genes were identified. Among these, sixteen genes except NEFH, HOXA9, and CLDN5 have not been reported previously using the conventional DNA methylation inhibitors. Methylation status of two genes, SLC32A1 and CSMD2, were further analyzed by methylation-specific PCR and found that SLC32A1 and CSMD2 were methylated in 100% (21/21) and 83% (15/18) pancreatic cancer cell lines analyzed, respectively. Our results suggest that “MeTA” is a highly efficient method to isolate methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer. Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control) were transfected with pcDNA6/myc-His vector or pcDNA6-3xFLAG-NFkB (AD)-MBD and were harvested 48 h after transfection.

表观遗传沉默基因的鉴定及特征分析，在癌症研究中具有极高的应用价值。尤为关键的是，高甲基化基因的相关信息可为解析表观遗传在肿瘤发生中的作用提供线索；而以肿瘤特异性方式频繁发生甲基化的基因，则可作为肿瘤标志物加以应用。此前，科研人员广泛使用5-氮杂胞苷（5-aza-cytidine）、5-氮杂-2’-脱氧胞苷（5-aza-2’-deoxycytidine）等DNA甲基化抑制剂，来筛选表观遗传沉默基因。但这类抑制剂常会上调启动子未发生甲基化的基因，特异性欠佳。为提升癌症中此类甲基化介导的沉默基因的检测特异性与灵敏度，本团队尝试优化检测手段，并成功开发出一种名为“甲基CpG靶向转录激活（methyl-CpG targeted transcriptional activation, MeTA）”的新方法：该方法借助带有甲基CpG结合域（methyl-CpG binding domain, MBD）的转录激活片段，可特异性识别并结合甲基化DNA。由于MBD蛋白实际可介导癌症中与启动子高甲基化相关的抑癌基因的转录沉默，因此MeTA被认为是筛选此类基因的理想方法之一。在本研究中，我们将MeTA方法应用于3株代表性胰腺癌细胞系：AsPC-1、MIA PaCa-2及PANC-1，并以正常胰腺导管上皮细胞系HPDE作为对照。此前已有研究通过5-氮杂-2’-脱氧胞苷对上述所有细胞系的表达谱进行过分析。首先，我们通过逆转录聚合酶链反应结合Southern印迹杂交，对NEFH、NPTX2、SFRP1、TIMP3及UCHL1这5个基因的表达进行了分析；上述基因已知在至少1株上述癌细胞系中发生了甲基化。结果证实，MeTA可使所有这些基因的表达均出现上调。随后，我们筛选了经MeTA处理后，在3株癌细胞系中表达上调至少2倍的基因，共鉴定得到19个符合条件的上调基因。其中，除NEFH、HOXA9及CLDN5之外的16个基因，此前尚未见通过传统DNA甲基化抑制剂筛选得到的报道。我们进一步通过甲基化特异性PCR（methylation-specific PCR）分析了SLC32A1与CSMD2两个基因的甲基化状态，结果显示：在所分析的胰腺癌细胞系中，SLC32A1的甲基化率为100%（21/21），CSMD2的甲基化率为83%（15/18）。本研究结果表明，MeTA是一种高效的人类胰腺癌甲基化介导转录沉默基因分离方法，且该方法可推广应用于其他类型的人类癌症研究。本研究将3株代表性胰腺癌细胞系AsPC-1、MIA PaCa-2、PANC-1及正常胰腺导管上皮细胞系HPDE（作为对照）分别转染pcDNA6/myc-His空载体与pcDNA6-3xFLAG-NFkB（AD）-MBD重组载体，并于转染48小时后收集细胞。