5-氮杂脱氧胞苷对肝癌细胞HepG2中抑癌基因FHIT表达的影响

Effect of 5-Aza-dC on FHIT gene expression in hepatocellular carcinoma cell line HepG2

背景与目的:肝癌细胞中由于FHIT基因过度甲基化而导致其表达降低。本实验应用甲基化酶抑制剂5-氮杂脱氧胞苷(5-Aza-2'-deoxycytidine,5-Aza-dC)作用肝癌细胞株HepG2,观察用药后肝癌细胞中FHIT mRNA和蛋白表达的变化,并观察5-Aza-dC对细胞增殖的影响。方法:以5-Aza-dC作用HepG2细胞,采用甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction,MSP)检测HepG2细胞中FHIT甲基化变化;采用RT-PCR检测FHIT mRNA的表达;采用细胞免疫组织化学染色和Western blot方法检测FHIT蛋白表达,MTT法检测HepG2细胞增殖情况。结果:5-Aza-dC处理前,HepG2细胞FHIT基因呈甲基化状态,mRNA及蛋白表达缺失;经5-Aza-dC作用后,MSP显示HepG2细胞FHIT基因甲基化逆转;经1.0μmol/L、2.0μmol/L及4.0μmol/L的5-Aza-dC作用48h后,FHIT基因mRNA扩增出产物的吸光度值分别为0.80±0.32、1.41±0.54和1.51±0.61,Western blot检测产物积分灰度值分别为0.33±0.20、1.00±0.26和1.12±0.38;当药物浓度达到1.0μmol/L时出现了对HepG2细胞增殖的抑制作用。结论:5-Aza-dC能明显逆转HepG2细胞的FHIT基因异常甲基化,激活因高甲基化所致基因沉默的再转录,诱导该基因的表达;同时抑制细胞增殖。

Background and Objective： FHIT gene methylation leads to down- regulation of its expression in hepatocellular carcinoma （HCC） cells. This study was to detect the expression of FHIT mRNA and protein in HCC cell line HepG2 after treatment of methylase inhibitor 5-Aza-2＇-deoxycytidine （5-Aza-dC）, and observe the effect of 5-Aza-dC on the proliferation of HepG2 cells. Methods： HepG2 cells were treated with 5-Aza-dC. Methylation of FHIT in HepG2 cells was detected by methylation-specific polymerase chain reaction （MSP）. FHIT mRNA expression was detected by reverse transcription-polymerase chain reaction （RT-PCR）. FHIT protein expression was detected by immunohistochemistry and Western blot. Cell proliferation was detected by MTT assay. Results： Before treatment of 5-Aza-dC, FHIT gene methylation was detected in HepG2 cells, while no FHIT mRNA and protein expression was detected. The hypermethylation of FHIT gene in HepG2 cells was effectively reversed after treatment of 5-Aza-dC. When HepG2 cells were treated with 1.0, 2.0, and 4.0 μmol/L of 5-Aza-dC for 48 h, the mRNA levels of FHIT were 0.80±0.32, 1.41±0.54, and 1.51±0.61, respectively; the protein levels of FHIT were 0.33±0.20, 1.00±0.26, and 1.12± 0.38, respectively. Cell proliferation was significantly inhibited after being treated with 5-Aza-dC. Conclusion： 5-Aza-dC can reverse the abnormal methylation of FHIT gene, activate the silenced gene and induce FHIT mRNA and protein expression in HepG2 cells.