纳米二氧化硅对HaCaT细胞中PAPR-1基因表达及其启动子甲基化的影响

Effect of silicon dioxide nanoparticles on expression and DNA methylation of PARP-1 gene in HaCaT cells

目的研究纳米二氧化硅（nm—SiO2）对人皮肤表皮细胞系（HaCaT）细胞中聚腺苷二磷酸核糖聚合酶-1（poly[ADP-ribose]polymerase 1，PARP-1）基因的表达及其基因启动子区甲基化的影响。方法分别以2．5、5．0、10．0μg／ml的nm-SiO2溶液和10μg／ml的微米级SiO2（micro-SiO2）溶液处理HaCaT细胞24h；以3μmol／LDNA甲基化转移酶抑制剂5-氮杂-2-脱氧胞苷（5-aza—CdR，DAC）处理48h10μg／ml的HaCaT细胞为阳性对照（DAC组），并设立溶剂对照组（CTRL组）。应用荧光定量聚合酶链式反应（polymerase chain reaction，PCR）和蛋白质印迹法（Western-blot法）检测PARP-1在mRNA水平和蛋白水平的表达变化，应用亚硫酸氢盐测序（bisulfite pyrosequence，BSP）法检测PARP-1基因启动子区CpG岛的甲基化状态。结果HaCaT细胞暴露nm-SiO224h后，经nm-SiO2处理后，micro-SiO2染毒组和2．5μg／ml染毒组PARP-1表达与CTRL组的差异无统计学意义（P〉0．05），与CTRL组比较，5、10μg／ml染毒组及DAC处理组细胞的PARP-1表达下调，差异有统计学意义（P〈0．05），且其下调水平随着浓度的增大而增高。BSP结果显示PARP-1启动子区-229点甲基化状态升高。结论nm-SiO2可以降低HaCaT细胞中PARP-1基因的表达，可能与其对PARP-1基因启动子区CpG岛的甲基化影响有关。

Objective To study the effect of silicon dioxide nanoparticles on the expression and promoter region CpG islands methylation of （Poly [ADP-ribose] polymerase 1, PARP-1 ） gene in human HaCaT Cell. Methods HaCaT Cells were treated with nm-SiO2 at 0, 2.5, 5 and 10 μg/mL and micro-SiO2 at 10 μg/ml for 24 h and DAC treatment was given at 10 μg/ml group for 48 h. Real-time PCR and western blot assay was used to detect the expression of PARP-1 mRNA and protein. BSP （Bisulfite Pyrosequence, BSP）assay was used to detect the promoter region CpG islands methylation status of PARP-1 gene. Results After exposure to nano- SiO2 particles, compared to CTRL group, the mRNA and protein expression of PARP-1 in micro-SlOE and 2.5 μg/ml group unchanged, but he mRNA and protein expression of PARP-1 in 5,10 μg/ml as well as DAC group was down-regulated and there are statistical significance between CTRL group and 5,10 μg/ml as well as DAC group and the PARP-1 promoter region CpG islands showed methylation. Conclusion nano-SiO2 can downregulate PARP-1 expression in HaCaT Cell and this is associated with the change in the methylafion of PARP-1 gene promoter region CpG islands induced by nano-SiO2 particles.