目的 了解亚砷酸钠(NaAsO2)对人皮肤角质形成细胞(HaCaT细胞)组蛋白H3第36位赖氨酸三甲基化(H3K36me3)修饰水平、O6甲基鸟嘌呤DNA甲基转移酶(MGMT)基因mRNA转录水平的影响,探讨H3K36me3调控MGMT基因转录与砷致DNA损伤的关系,为砷中毒预防和治疗提供新思路和科学依据.方法 1.25、2.50、5.00、10.00 μmol/L NaAsO2处理HaCaT细胞24 h,10.00 μmol/LNaAsO2处理HaCaT细胞6、12、24h,剂量以0.00μmol/L、时间以0h为对照组.采用单细胞凝胶电泳法检测HaCaT细胞DNA损伤情况;免疫印迹法检测H3K36me3总体修饰水平;实时荧光定量PCR检测MGMT基因mRNA表达水平;定量染色质免疫沉淀技术检测MGMT基因编码区(ChIP1、ChIP2区域)H3K36me3修饰水平.结果 ①DNA损伤检测结果:2.50、5.00、10.00 μmol/L染砷组HaCaT细胞彗星尾部DNA百分含量(Tail DNA％,11.83±1.15、16.85±2.52、24.23±2.75)、彗星尾矩(Olive tail moment,OTM,10.90±1.13、16.19±2.26、23.83±2.79)明显高于对照组(0.00 μmol/L,2.40±0.51、2.26±0.40,P均＜0.05);10.00 μmol/L NaAsO2处理HaCaT细胞12、24h,TailDNA％(15.51±1.92、24.18±2.42)、OTM(13.58±2.04、23.14±2.11)明显高于对照组(0 h,3.66±1.02、3.38±1.00,P均＜0.05).②H3K36me3总体修饰水平检测结果:与对照组(0.00μmol/L,100.00±0.00)比较,2.50、5.00、10.00μmol/L染砷组H3K36me3总体修饰水平(60.59±9.75、57.82±11.28、39.45±7.09)降低(P均＜0.05);与对照组(0 h,100.00±0.00)比较,12、24 h染砷组H3K36me3总体修饰水平(48.47±9.67、47.75±6.98)亦降低(P均＜0.05).③不同剂量染砷组HaCaT细胞H3K36me3总体修饰水平与Tail DNA％、OTM均呈负相关关系(r=-0.897、-0.903,P均＜0.05).④MGMT基因mRNA表达水平检测结果:与对照组(0.00 μmol/L,100.00±0.00)比较,2.50、5.00、10.00μmol/L染砷组MGMT基因mRNA表达水平(78.20±3.50、61.40±2.60、49.15±4.70)降低(P均＜0.05).⑤MGMT基因编码区H3K36me3修饰水平检测结果:各剂量染砷组MGMT基因编码区ChIP1、ChIP2区域未观察到组蛋白H3K36me3的富集规律(P均＞0.05).结论 砷诱导的HaCaT细胞DNA损伤可能受H3K36me3的调控;砷可导致HaCaT细胞MGMT基因mRNA转录抑制,但H3K36me3调控MGMT基因mRNA转录抑制与砷致DNA损伤关系不密切.%Objective To observe the influences of NaAsO2 on H3K36me3 modifications,mRNA transcription of O6-methylguanine-DNA methyltransferase gene (MGMT) in HaCaT cells,and to explore the relationship between the transcription of MGMT gene regulated by H3K36me3 and DNA damage induced by arsenic,in order to provide new ideas and scientific basis for prevention and intervention of arsenism.Methods HaCaT cells were treated with 1.25,2.50,5.00 and 10.00 μmol/L NaAsO2 for 24 h,and were also treated with 10.00 μmol/L NaAsO2 for 6,12 and 24 h.HaCaT cells that treated with 0.00 pmol/L NaAsO2 and 0 h were used as blank control group.The degree of DNA damage in peripheral blood cells was detected by single cell gel electrophoresis.The level of H3K36me3 modifications was detected using Western blotting.Quantitative real-time polymerase chain reaction was used to detect the mRNA levels of MGMT gene.Quantitative chromatin immuno-precipitation was used to detect the level of H3K36me3 modifications in the coding regions (ChIP1 and ChIP2) of MGMT gene.Results ①Among the groups of HaCaT cells treated with 2.50,5.00 and 10.00 μmol/L NaAsO2,the levels of tail DNA％ (11.83 ± 1.15,16.85 ± 2.52,24.23 ± 2.75) and olive tail moment (OTM,10.90 ± 1.13,16.19 ± 2.26,23.83 ± 2.79)were significantly increased compared with those of the control group (0.00 μmol/L,2.40 ± 0.51,2.26 ± 0.40,all P ＜ 0.05).After treated with 10.00 μmoFL NaAsO2 for 12 and 24 h,compared with the control group (0 h,3.66 ± 1.02,3.38 ± 1.00),the degrees of tail DNA％ (15.51 ± 1.92,24.18 ± 2.42) and OTM (13.58 ± 2.04,23.14 ± 2.11)were significantly increased (all P ＜ 0.05).②Compared with the control group (0.00 μmol/L,100.00 ± 0.00),the levels of H3K36me3 modifications (60.59 ± 9.75,57.82 ± 11.28,39.45 ± 7.09) were lower at the dosages of 2.50,5.00 and 10.00 μmol/L NaAsO2 (all P ＜ 0.05).Compared with the control group (0 h,100.00 ± 0.00),the levels of H3K36me3 modifications (48.47 ± 9.67,47.75 ± 6.98) were lower after treated with 10.00 μ mol/L NaAsO2 for 12 and 24 h (all P ＜ 0.05).③The levels of H3K36me3 modifications in HaCaT cells exposed to different doses of NaAsO2 were negatively associated with the tail DNA％ and OTM (r =-0.897,-0.903,all P ＜ 0.05).④Compared with the control group (0.00 μmol/L,100.00 ± 0.00),the mRNA levels of MGMT gene were lower at the dosages of 2.50,5.00 and 10.00 pmol/L NaAsO2 (78.20 ± 3.50,61.40 ± 2.60,49.15 ± 4.70,all P ＜ 0.05).⑤There was no observed H3K36me3 enrichmem regularity in the gene encoding ChIP1 and ChIP2 regions of MGMT gene in all doses of NaAsO2 groups (all P ＞ 0.05).Conclusions H3K36me3 may be involved in the regulation of arsenicinduced DNA damage in HaCaT cell.Amenic could inhibit the mRNA transcription of MGMT gene in HaCaT cells,but the transcription of MGMT gene regulate by H3K36me3 is not closely related to DNA damage induced by arsenic.
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