正常上皮细胞特异性-1基因超甲基化在肝癌发病机制中的作用

Normal epithelial cell specific-1 gene hypermethylation in the carcinogenesis of hepatocellular carci-noma

目的：探讨正常上皮细胞特异性-1基因在正常肝细胞和肝癌细胞株中的表达及其甲基化状态对该基因表达和细胞生物学的影响。方法用实时PCR 和实时 QPCR 方法检测NES1 mRNA在肝癌细胞株HepG2和正常肝细胞L02中的表达，用MSP检测基因甲基化状态。以不同浓度DNA甲基化酶抑制剂（5-aza-dC）处理肝癌细胞株后，分别用实时QPCR检测NES1 mRNA表达量， MTT法检测细胞的存活率，蛋白印迹（ WB）检测细胞周期蛋白cyclin D1、P21和P53表达水平。结果NES1 mRNA在肝癌细胞株中的表达量较正常肝细胞明显降低。肝癌细胞株中NES1外显子3 CpG岛的甲基化程度远远高于正常肝细胞。肝癌细胞株去甲基化后，NES1 mRNA表达水平有所上调，细胞存活率明显下降，P21和P53表达水平上升，cyclinD1表达水平下调。结论 NES1在肝癌细胞株中的低表达与该基因外显子3 CpG岛甲基化有关。 NES1基因外显子3甲基化可能是肝癌细胞中NES1表达缺失的分子机制。去甲基化药物5-aza-dC可通过阻滞细胞周期而抑制肝癌细胞株的增殖。

Objective To probe the gene expression of normal epithelial cell specific-1 （NES1） in normal liver cells and liver cancer cell lines , and investigate the gene methylation status and its impacts on gene expression and cell biology .Methods The expression level of NES1 mRNA was detected in HepG2 and L02 cells by RT-PCR and RT-QPCR, and the level of gene methylation was examined by MSP .We de-tected cell viability by MTT , NES1 mRNA by RT-QPCR and cyclinD1, P21 and P53 level by Western blot after treating cells with 5-Aza-CdR.Results Compared with L02, NES1 mRNA expression in HepG2 was significantly reduced, and the level of NES1 exon 3 CpG island methylation in HepG2 cells was much higher than that in L02 cells.After demethylation , NES1 mRNA expression and protein level of p 21 and p53 in HepG2 cells were up-regulated , while the cell viability and the level of CyclinD 1 were decreased .Conclu-sions In hepatocellular carcinoma , low expression of NES1 mRNA is related to the gene exon 3 CpG island methylation .NES1 exon 3 methylation may be one of the molecular mechanisms for reducing NES 1 mRNA level, and 5-aza-dC could inhibit cell proliferation by inducing cell cycle arrest in HepG 2 cells.