5氮杂脱氧胞苷对鼻咽癌细胞RASSF1A基因甲基化和mRNA表达的影响

目的:研究5氮杂脱氧胞苷对鼻咽癌CNE2细胞中RASSF1A基因甲基化和mRNA表达的影响。方法:用不同浓度(0、5、10、20μmol/L)5氮杂脱氧胞苷处理CNE2细胞,采用甲基化特异性PCR(MSP)法对处理后细胞中RASSF1A基因甲基化状态进行检测;并用SYBR Green qReal Time-PCR法检测RASSF1A mRNA的表达。结果:阴性对照组CNE2细胞中,RASSF1A基因呈完全甲基化状态,当用5μmol/L 5氮杂脱氧胞苷处理后,CNE2细胞出现非甲基化产物,20μmol/L 5氮杂脱氧胞苷处理后,CNE2细胞甲基化状态完全被逆转,均为非甲基化产物。阴性对照组CNE2细胞RASSF1A基因呈低表达,经5～20μmol/L 5氮杂脱氧胞苷处理后,RASSF1A mRNA的相对表达量逐渐增加,10和20μmol/L 5氮杂脱氧胞苷处理组,RASSF1A mRNA表达水平均明显高于阴性对照组(P0.01);且20μmol/L处理组明显高于5μmol/L处理组(P0.01)。结论:CNE2细胞RASSF1A基因甲基化可以被5氮杂脱氧胞苷逆转,且5氮杂脱氧胞苷可促进RASSF1A mRNA的表达。

Effect of 5-Aza-CdR on methylation and mRNA expression of RASSF1A gene in nasopharyngeal carcinoma cells

OBJECTIVE：The relationship between methylation of RASSF1A gene and mRNA expression in nasopharyngeal carcinoma cell line CNE2 was investigated.METHODS：CNE2 cells were treated with various concentrations of 5-Aza-CdR.Methylation of RASSF1A gene was evaluated by methylation—specific polymerase chain reaction（MSP）.Expression of RASSF1A mRNA was detected by SYBR Green qRealTime-PCR.RESULTS：RASSF1A gene was completely methylated in the negative control group cell.De-methylation occurred in the 5μmol/L of 5-Aza-CdR. Methylation was completely reversed by 20μmol/L of 5-Aza-CdR.The expression of RASSF1A mRNA in the negative control group was low.The expression level increased gradually by 5,10 and 20μmol/L 5-Aza-CdR treatment. The levels in 10 and 20μmol/L treated group were significantly improved in comparison with the negative control group （P〈0.01）.RASSF1A mRNA in 20μmol/L treated group was obviously higher than that in 5μmol/L treated group （P〈0.01）.CONCLUSION：Methylation of RASSF1A gene in the nasopharyngeal carcinoma cell line CNE2 could be reversed by 5-Aza-CdR,promoting the expression of RASSF1A gene mRNA.