黑色素瘤相关抗原A1在肝癌细胞株中的表达与基因甲基化程度的相关性

目的 探讨人肝癌细胞株中黑色素瘤相关抗原A1(MAGE-A1)表达状况与肝癌细胞基因甲基化的关系。方法 提取10种人肝癌细胞株的总RNA,用逆转录聚合酶链反应对细胞株的MAGE-A1基因的表达进行检测;提取10种人肝癌细胞株的基因组DNA,用限制性酶切和Southern印迹杂交对每种细胞的基因组甲基化程度进行定量分析。另外对基因组DNA行HpaⅡ酶切后,用引物CDS21、EDP4和CDS20、EDP4进行聚合酶链反应扩增,然后用特异性探针杂交来检测肝癌细胞株MAGE-A1启动子的甲基化状态。用特异性序列聚合酶链反应方法检测每一种细胞株的人白细胞抗原-A位点型别。结果 QGY-7703、SMMC-7721、HLE、BEL-7402、BEL-7404、BEL-7405肝癌细胞株的MAGE-A1基因表达阳性,且细胞分化程度均为中低分化;HepG2 2．2．15、HepG2、QGY-7701和Huh7肝癌细胞株的MAGE-A1基因表达阴性,且细胞分化程度均为高中分化。通过定量分析表明,MAGE-A1表达的肝癌细胞株与MAGE-A1不表达的肝癌细胞株比较,前者基因组甲基化程度较低(t=2．896,P=0．02)。肝癌细胞株MAGE-A1基因启动子区甲基化分析结果表明HepG2 2．2．15、HepG2、QGY-7701和Huh7为高甲基化,SMMC-7721、HLE、BEL-7402、BEL-7404、BEL-7405为低甲基化。结论 肝癌细胞株MAGE-A1 mRNA表达与其基因甲基化程度有关。

Expression of MAGE-A1 in human hepatoma cell lines associated with genic hypomethylation

Objective To investigate the correlation between MAGE-Al mRNA expression and genie demethylation in hepatoma cell lines. Methods Total RNA and genomic DNA were prepared from 10 human hepatoma cell lines. MAGE-1 mRNA expression was determined with RT-PCR and the level of genome-wide demethylation was evaluated by enzyme digestion and Southern blot assay. The genomic DNA was digested by Hpall, then the promoter of MAGE-A1 gene was amplified with primers CDS21, EDP4 and CDS20. EDP4 and the PCR products were further hybridized with a probe to detect the methylation in the promoter of the MAGE-A1 gene. HLA-A locus was typed using SSP kit. Results In cell lines QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404 and BEL-7405, MAGE-A1 mRNA expression was positive and cell differentiation was moderate or low. In cell lines of HepG2 2.2.15, HepG2, QGY-7701 and Huh7, MAGE-A1 mRNA expression was negative and cell differentiation was well or moderate. The level of genomic demethylation in MAGE-A1 mRNA positive cell lines was much higher than that in MAGE-A1 mRNA negative cell lines (t = 2.896, P = 0.02). The methylation analysis showed that methylation in the promoters of MAGE-A1 gene of HepG2 2.2.15, HepG2, QGY-7701 and Huh7 was high, and that methylation in those of SMMC-7721, HLE, BEL-7402, BEL-7404, and BEL-7405 was low. Conclusion The results suggest that MAGE-A1 mRNA expression in the human hepatoma cell lines is associated with genie hypomethylation.