H3K36me3调控O^6-甲基鸟嘌呤DNA甲基转移酶基因与砷致人皮肤角质形成细胞DNA损伤的关系  

Relationship between O^6-methylguanine-DNA methyltransferase gene regulated by H3K36me3 and DNA damage induced by arsenic in HaCaT Cells

作　　者：李军 马璐[1] 谢琅[1] 陈丽[1] 张爱华[1] 

机构地区：[1]贵州医科大学环境污染与疾病监控教育部重点实验室,贵州医科大学公共卫生学院,贵阳550025

出　　处：《中华地方病学杂志》2017年第2期107-112,共6页Chinese Journal of Endemiology

基　　金：国家自然科学基金（81360411、81430077）

摘　　要：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、O^6-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、10．00μmol／L NaAsO2处理HaCaT细胞24h，10．00μmol／L NaAsO2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、10．00μmol／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（0．00μmoL／L，2．40±0．51、2．26±0．40，P均〈0．05）；10．00μmoL／L NaAsO2处理HaCaT细胞12、24h，Tail DNA％（15．51±1．92、24．18±2．42）、OTM（13．58±2．04、23．14±2．11）明显高于对照组（0h，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（0h，100．00±0．00）比较，12、24h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�Objective To observe the influences of NaAsO2 on H3K36me3 modifications, mRNA transcription of O^6-methylguaninc-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 μmol/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 ehromatin immuno-precipitation was used to deteet the level of H3K36me3 modifications in the coding regions （CHIP1 and CHIP2） of MGMT gene. Results （1）Among 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 μmol/L NaAsO2 for 12 and 24 h, compared with the eontrol 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）. （2）Compared 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 le

关 键 词：亚砷酸盐类 组蛋白类 O^6-甲基鸟嘌呤DNA甲基转移酶 DNA损伤 

分 类 号：R599.1[医药卫生—内科学]