氢醌HQ对K562细胞WRN基因甲基化水平及DNMT1表达的影响

Effects of hydroquinone on methylation level of WRN gene and expression of DNMT1 in K562 cells

摘要:

目的：探讨氢醌（hydroquinone，HQ）对K562细胞中WRN基因甲基化水平及DNMT1表达的影响。方法：K562细胞生长至对数期后，设置空白对照组，低（15 μmol/L）、中（30 μmol/L）、高（60 μmol/L）剂量HQ组，重复间隔染毒72 h，空白对照组加入等量的PBS培养。MTT比色法检测细胞存活率；重亚硫酸盐测序法（bisulfite sequencing PCR，BSP）检测细胞WRN基因甲基化水平；FQ-PCR法检测DNMT1基因的mRNA表达情况；免疫印迹法检测DNMT1基因的蛋白表达情况。结果：①MTT结果显示，细胞存活率随着HQ染毒浓度增加而下降（P=0.000）。②BSP检测结果显示，低、中、高剂量HQ组甲基化率与对照组比较，差异有统计学意义（χ2=7.50，P=0.048）。③与对照组比较，低、中、高剂量HQ组DNMT1基因mRNA及蛋白表达量呈下降趋势（P=0.000）。结论：HQ染毒K562细胞可引起WRN基因甲基化水平增高，同时下调DNMT1基因的表达。

Abstract:

Objective：To explore the influences of hydroquinone（HQ） on methylation level of WRN gene and expression of DNMT1 gene in K562 cells. Methods：The low dose HQ group，middle dose HQ group andhigh dose HQ group were treated with 15 μmol/L，30 μmol/L，60 μmol/L hydroquinone for 72h at intervals and repeatedly，while the blank control group cells were cultured with PBS. The survival rate of each experimental group was detected by MTT assay. The methylation level of WRN gene was detected by bisulfite sequencing PCR（BSP）. The protein expression of DNMT1 gene was detected by Western blot. The mRNA expressions of DNMT1 gene were detected by real-time fluorescence quantitative PCR. Results：①The results of MTT showed that the cell viability decreased with the increasing of HQ concentration（P=0.000）. ②Compared with the that of control group，the difference of the methylation rates of low dose HQ group，middle dose HQ group，high dose HQ group，was statistically significant（ χ2=7.50，P=0.048）. ③Compared with that of the control group，the relative expression of mRNA and protein of DNMT1 of each HQ group decreased（P=0.000）. Conclusion：HQ can increase the methylation level of WRN gene and down-regulated the expression of DNMT1 gene in K562 cells.