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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
Objective To investigate the relationship between the expression of MAGE-A1 and the methylation of hepatocellular carcinoma cells in human hepatocellular carcinoma cell lines. Methods The total RNA of 10 human hepatocellular carcinoma cell lines was extracted and the expression of MAGE-A1 gene was detected by RT-PCR. The genomic DNA of 10 human hepatocellular carcinoma cell lines was extracted and digested with restriction endonucleases Southern The degree of genomic methylation in each cell was quantitatively analyzed by blotting. In addition, the genomic DNA was digested with HpaII and amplified by polymerase chain reaction using primers CDS21, EDP4, CDS20 and EDP4, and then the methylation status of the MAGE-A1 promoter was detected by specific probe hybridization. Human leukocyte antigen A site type was determined for each of the cell lines by a specific sequence polymerase chain reaction method. Results The expression of MAGE-A1 gene in QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404 and BEL-7405 hepatocellular carcinoma cell lines was positive and the degree of cell differentiation was moderately differentiated. HepG2 2.2.15, HepG2, MAGE-A1 gene expression in QGY-7701 and Huh7 hepatoma cell lines was negative, and the degree of cell differentiation was high differentiation. Quantitative analysis showed that the former had a lower degree of methylation (t = 2.896, P = 0.02) compared with the MAGE-A1 non-expressing hepatocellular carcinoma cell line. The results of methylation analysis of promoter region of MAGE-A1 gene in Hepatocellular carcinoma cell line showed that HepG2 2.2.15, HepG2, QGY-7701 and Huh7 were hypermethylated and SMMC-7721, HLE, BEL-7402, BEL-7404 and BEL-7405 were Hypomethylation. Conclusion The expression of MAGE-A1 mRNA in hepatocellular carcinoma cell line is related to the gene methylation