正常上皮细胞特异性-1基因在胃癌细胞株中的表达及甲基化调控

Epigenetic modification regulates the expression of NES1 gene in human gastric cancer cell lines

目的:研究正常上皮细胞特异性-1(NES1)基因表达及其CpG岛甲基化状况与胃癌的关系。方法:分别培养胃癌细胞MKN-28(高分化)、SGC-7901、AGS(均中分化)、MKN-45(低分化)、HGC-27(未分化)和原代正常胃上皮细胞。提取细胞RNA,经RT-PCR检测NES1在各细胞株中的表达。以不同浓度DNA去甲基化抑制剂5-aza-dc处理胃癌细胞,RT-PCR检测其NES1mRNA表达。提取细胞基因组DNA,甲基化修饰后MSP分析其CpG岛甲基化状态。结果:各肿瘤细胞株中NES1mRNA表达较胃正常上皮细胞有不同程度降低。甲基化检测显示NES1表达减少的胃癌细胞株均存在不同程度的NES1基因外显子3CpG岛甲基化。NES1表达降低的胃癌细胞株经5-aza-d处理后NES1表达上调。结论:NES1胃癌细胞株中低表达,表达下调与该基因外显子3周围CpG岛甲基化有关。5-aza-dc可使NES1表达降低的胃癌细胞株重新表达NES1mRNA。提示NES1基因CpG岛甲基化可能是NES1在胃癌细胞系中表达缺失的分子机制。

Objective To investigate the normal epithelial cell specific-1 （NES1） gene expression and the methylation of NES1 gene CpG island in gastric cancer. Methods Five gastric cancer cell lines, including MKN-28, SGC-7901, AGS, MKN-45 and HGC-27, and human gastric epithelial cells were cuhured.The NES1 mRNA expression was detected by RT-PCR. Gastric cancer cell lines were treated with DNA methyhransferase inhibitor, 5-aza-2＇-deoxycytidine （5-aza-dc）. And then the NES1 gene expression was detected using RT-PCR. The DNA methylation status of NES1 gene was assayed by methylation-specific PCR（MSP）. Results In comparison with the human gastric epithelial cells, the NES1 mRNA expression decreased in the gastric cancer cell lines. Through the MSP, the aberrant methylation of NES1 gene in the exon 3 was found. It showed a strong correlation between the exon 3 hypermethylation and the loss of NES1 mRNA expression in the gastric cancer cell lines. After the 5-aza-dc treatment, the NES1 expression in the NES1-nonexpressing tumor cell lines was up-regulated. Conclusions Hypermethylation is responsible for the tumor-specific loss of NES1 gene expression in the gastric cancer cells. Treatment with a demethylating agent can lead to the reexpression of NES1 in the gastric cancer cell lines. It suggests an important role of the hypermethylation in the loss of NES1 expression.