5-氮杂-2'-脱氧胞苷和曲古抑酶素A对亚砷酸钠处理细胞中DNMT1和HDAC1基因转录及表达的影响

Effects of 5-AZA-dC and TSA on transcription and expression of DNA methyltransferase 1(DNMT1) and histone deacetylase 1(HDAC1) gene in NaAsO_2-treated cells

目的探讨5-氮杂-2'-脱氧胞苷(5-AZA-dC)和曲古抑酶素A(TSA)对亚砷酸钠(NaAsO2)处理的人皮肤角质形成细胞(HaCaT细胞)中DNA甲基转移酶1(DNMT1)、组蛋白去乙酰化酶1(HDAC1)基因mRNA转录和蛋白表达的影响。方法以25μmol/L NaAsO2重复间隔染毒HaCaT细胞72 h(25μmol/L NaAsO2处理24 h,隔天再次进行相同处理,共处理3次),再分别以1.5、3.0、6.0μmol/L 5-AZA-dC作用于NaAsO2处理细胞72 h,125、250、500 nmol/L TSA作用于NaAsO2处理细胞48 h,以实时荧光定量PCR(Q-PCR)技术检测DNMT1及HDAC1基因的mRNA转录,以免疫印迹分析(western blotting)检测DNMT1及HDAC1蛋白表达。设不进行任何处理的细胞为空白对照,仅染砷的细胞为阳性对照。结果 1.5、3.0、6.0μmol/L 5-AZA-dC作用于NaAsO2处理细胞后,DNMT1及HDAC1 mRNA转录相对表达量与仅染砷的阳性对照比较,差异无统计学意义;DNMT1蛋白相对表达量降低,与仅染砷的阳性对照比较,差异有统计学意义(P<0.05);HDAC1蛋白相对表达量与仅染砷的阳性对照比较,差异无统计学意义。125、250、500 nmol/L TSA作用于NaAsO2处理细胞后,DNMT1及HDAC1 mRNA转录相对表达量与仅染砷的阳性对照比较,差异无统计学意义;DNMT1及HDAC1蛋白相对表达量降低,与仅染砷的阳性对照比较,差异有统计学意义(P<0.05)。结论 5-AZA-dC能抑制砷所致的DNMT1蛋白高表达,TSA能抑制砷所致的DNMT1及HDAC1蛋白高表达,这为进一步探索砷中毒的表观遗传学治疗提供了科学依据。

Objective To observe the influences of 5-AZA-dC and TSA on mRNA transcription and protein expression of DNA methyhransferase 1 （DNMT1） and histone deacetylase 1 （HDAC1） gene in NaAsO2-treated HaCaT cells. Methods HaCaT cells were treated for 72 hours at intervals and repeatedly by 25 μmol/L NaAsO2, then the cells were exerted respectively to 1.5, 3.0 and 6.0 μmol/L DNA methytransferases inhibitor （5-AZA-dC） for 72 hours and 125, 250 and 500 nmol/L histonedeacetylase inhibitor （TSA） for 48 hours. The mRNA transcription of DNMT1 and HDAC1 was detected by real-time quantitative PCR, the protein expression of DNMT1 and HDA C1 was detected by Western blot. HaCaT cells without any treatment were set as the blank control, HaCaT cells treated by arsenic only were set as the positive control. Results Among the groups of NaAsO2-treated cells exerted by 1.5, 3.0, 6.0 μmol/L 5-AZA-dC, compared with the positive control, the differences of DNMT1 and HDAC1 mRNA transcription were not significant; the level of DNMT1 protein expression decreased significantly compared with those of the positive control （P〈0.05）, the difference of HDAC1 protein expression were not significant. Among the groups of NaAsO2-treated ceils exerted by 125, 250 and 500 nmol/L TSA, compared with the positive control, the differences of DNMT1 and HDAC1 mRNA transcription were not significant, the level of DNMT1 and HDAC1 protein expression decreased significantly （P〈0.05）. Conclusion 5-AZA-dC can inhibit DNMT1 protein high expression induced by arsenic, TSA can inhibit DNMT1 and HDAC1 protein high expression induced by arsenic, which may provide scientific basis for exploring the epigenetic treatment of arsenism.