普鲁卡因与5'-氮杂-2'-脱氧胞苷对人肝肿瘤细胞株HepG2中Wif-1基因启动子甲基化影响的比较研究<
Comparison of the Effect Between Procaine and 5-aza-dc on Wnt Inhibitory Factor 1 Promoter Methylation of HepG2
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摘要: 目的 探讨普鲁卡因与5'-氮杂-2'-脱氧胞苷(5'-Aza-2'-deoxycycytidine,5-aza-dc)对人肝肿瘤细胞株HepG2经典Wnt信号通路抑制因子-1(Wif-1)基因启动子脱甲基化作用的影响。 方法 以普鲁卡因或5-aza-dc干预HepG2细胞,采用 RT-PCR及Western blot检测干预前后Wif-1基因mRNA及蛋白的表达;甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction,MSP)检测干预前后HepG2细胞中Wif-1甲基化变化。 结果 RT-PCR和Western blot结果显示经普鲁卡因或5-aza-dc作用前HepG2细胞Wif-1基因mRNA与蛋白呈低表达或缺失,经普鲁卡因或5-aza-dc作用后HepG2细胞Wif-1基因mRNA与蛋白呈高表达,各组间比较差异有统计学意义,且普鲁卡因实验组表达高于5-aza-dc实验组(P<0.05)。MSP结果显示,经普鲁卡因或5-aza-dc干预后HepG2细胞与干预前HepG2细胞比较均呈部分脱甲基化状态,对应的甲基化率分别为(14.41±3.37)%和(29.29±1.84)%(P<0.05)。 结论 普鲁卡因及5-aza-dc均可影响Wif-1基因启动子CpG岛的甲基化状态。普鲁卡因可以作为一种新的去甲基化药物且效果优于常规药物5-aza-dc。Abstract: Objective To observe the CpG island methylation status, mRNA and protein expression of the Wnt inhibitory factor-1 (Wif-1) gene with procaine or 5'-Aza-2'-deoxycycytidine (5-aza-dc) on HepG2. And to explore the comparison of the demethylation with 5-aza-dc or procaine. Methods HepG2 cells were treated with 5-aza-dc or procaine. Wif-1 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Wif-1 protein expression was detected by Western blot. The promoter methylation status of Wif-1 gene on treatment or not were detected by methylation-specific PCR (MSP). Results ① RT-PCR and Western blot exhibited that both Wif-1 mRNA and Wif-1 protein expression were positive in groups treated with procaine and 5-aza-dc and highly positive in the L0 cells group, but weakly even negative in the HepG2 cells without any treatment, the difference were statistically significant (P<0.05) (procaine experimental group was higher than that of 5-aza-dc experimental group, P<0.05). ② The positive rates of the promoter hypermethylated in procaine and 5-aza-dc groups were (14.41±3.37)% and (29.29±1.84)%. Both of the two groups showed a part of the de-methylation status (P<0.05). Conclusion Both of procaine and 5-aza-dc can reverse the abnormal methylation of Wif-1 gene. Procaine can be more effective than conventional used 5-aza-dc and could be a new demethylation drug.
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