短期暴露于纳米SiO2对HaCaT细胞基因组DNA甲基化的影响

Genome DNA hypomethylation in HaCaT cells after short exposure to SiO_2 nanoparticles

目的探讨纳米SiO2对体外培养细胞基因组总体DNA甲基化水平的影响。方法分别以2.5、5、10μg/ml纳米SiO2溶液和10μg/ml微米级SiO2处理人皮肤表皮细胞系(HaCaT)24h;以3μmol/L DNA甲基化转移酶抑制剂5-脱氧杂氮胞苷(DAC)处理48 h的10μg/ml组为阳性对照,并设立溶剂对照组。应用高效毛细管电泳(HPCE)定量分析纳米SiO2处理细胞24h后,基因组DNA总体甲基化的变化,用Q-PCR和Western Blot方法检测甲基化相关蛋白mRNA和蛋白的表达变化,并用DNA甲基化转移酶活性试剂盒检测DNA甲基化转移酶的活性。结果 HPCE定量分析结果显示纳米SiO2可引起HaCaT细胞总体甲基化程度降低,呈剂量依赖性关系;与正常细胞相比,微米SiO2以及2.5、5和10μg/ml纳米SiO2组分别降低37.8%、43.9%、54.9%和52.8%,差异有显著性(P<0.05);对照组5-aza-dC处理后使HaCaT细胞甲基化程度减少27.3%。各组酶的蛋白表达与mRNA表达水平及DNMTs活性变化具有相同的变化趋势,经纳米SiO2处理的HaCaT细胞,DNMT1、DNMT3a及MBD2表达随着纳米SiO2剂量的上升而下降,DAC组表达水平最低。结论纳米SiO2能引起HaCaT细胞整体基因组DNA甲基化水平降低,可能与DNA甲基化转移酶的酶活性降低有关。

Objective To observe the effect of SiO2 nanoparticles on genome DNA methylation profile in cultured ceils. Methods HaCaT cells were treated with nm-SiO2 at 2.5, 5 and 10μg/ml and micro-SiO2 at 10μg/ml for 24h and DAC treatment was given at 101μg/ml group for 48h. The mC/（mC ＋ C） percent was quantified by high performance capillary electrophoresis （HPCE） assay, and the expression level of mRNA and protein was detected by Real-time Q-PCR and westernblot assay. The activity of DNMTs was determined by DNA Methyltransferase Activity/Inhibition Assay Kit. Results HPCE assay showed that nm-SiO2-treated cells were decreased in some degree. An averageproportion of methylated mC/ （mC ＋ C） was 4.82% in control, 2.7% in 2.51μg/ml and 2. 17% in 10μg/ml groups, while 3.1% in micro-SiO2 groups, which got the consistent downtrend of genome methylation level during increasing nm-SiO2 dose nanoparticles. The mRNA expression level for DNMT1 decreased gradually with increased dose of nm-SiO2 nanoparticles. The alterations at protein level were similar to those at the mRNA level. Conclusion Genomic DNA methylation levels were decreased in HaCaT cells after short term exposure to SiO2 nanoparticles.