DNA去甲基化药物对肝癌SMMC-7721细胞株IGF2印迹状态及表达的影响

Effects of cytosine methylation inhibitor on imprinting and expression of insulin-like growth factor Ⅱ in hepatocellular carcinoma SMMC-7721 cell line

目的:研究5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对肝癌SMMC-7721细胞株IGF2印迹状态及表达的影响。方法:用终浓度为10μmol/L的5-氮杂-2′-脱氧胞苷处理培养的SMMC-7721细胞,不含5-Aza-CdR培养液培养的细胞作为对照组;IGF2基因的印迹状态使用PCR-RFLP方法检测;IGF2基因的表达使用半定量RT-PCR确定。结果:SMMC-7721细胞中IGF2基因处于母源印迹状态,为杂合子;5-氮杂-2′-脱氧胞苷处理后IGF2基因的mRNA表达量降低,电泳条带的半定量分析显示处理和对照之间的差异具有统计学意义(P<0.01),RFLP结果未见印迹丢失。结论:胞嘧啶去甲基化药物能够改变SMMC-7721细胞中IGF2的甲基化状态,降低IGF2mRNA的表达量,但并不引起IGF2基因完全失表达。

Objective：To investigate the effects of 5-aza-2′-deoxycytidine （5-Aza-CdR）on the imprinting status and expression of insulin-like growth factor Ⅱ （IGF2） in hepatocellular carcinoma cells.Methods：SMMC-7721 cells were cultured in the medium containing 10 μmol/L 5-Aza-CdR （final concentration）.The imprinting status of IGF2 was detected by polymerase chain reaction-restrictive fragment length polymorphism（PCR-RFLP） and the expression of IGF2 mRNA was determined by semi-quantitative duplex reverse transcription PCR （RT-PCR）.Results：Normal maternal imprints of IGF2 were found in SMMC-7721 cells （heterozygote）.5-Aza-CdR led to down-regulation of IGF2 transcripts,but failed to alter its imprinting status.The expression of mRNA analyzed by Gel-Pro Analyzer between the control and treatment showed significant statistical difference（P0.01）.Conclusion：The cytosine methylation inhibitor 5-Aza-CdR can result in demethylation of CpG island region and down-regulation in IGF2,but was incapable of causing loss of imprinting （LOI）.