亚砷酸钠对HaCaT细胞DNA甲基转移酶的影响及N-乙酰半胱氨酸的干预效应

韩进美; 黄红茜; 李凤敏; 张爱华; 韩雪

doi:10.13213/j.cnki.jeom.2020.19698

亚砷酸钠对HaCaT细胞DNA甲基转移酶的影响及N-乙酰半胱氨酸的干预效应

Effects of sodium arsenite on DNA methyl transferases and antagonistic effects of

摘要

摘要: 背景
砷暴露可致人皮肤病变，砷所致的DNA甲基化改变是其主要的致病机制之一，但砷如何引起DNA甲基化改变目前尚未明确。
目的
本研究以永生化的人皮肤角质形成细胞（HaCaT）为研究对象，检测亚砷酸钠（NaAsO₂）暴露对HaCaT细胞DNA甲基转移酶（DNMTs）mRNA和蛋白表达水平的影响，并探讨砷所致氧化应激与其所致DNMTs mRNA和蛋白表达水平改变之间的关联。
方法
给予HaCaT细胞不同浓度的NaAsO₂（0、0.625、1.25、2.50μmol·L^-1）处理，干预组给予10.0 μmol·L^-1抗氧化剂N-乙酰半胱氨酸（NAC）预处理4 h后加入2.50 μmol·L^-1 NaAsO₂，染毒时间为72 h。应用免疫荧光法检测细胞活性氧（ROS）含量，生化法检测细胞内丙二醛（MDA）含量，实时荧光定量PCR法检测细胞DNMT1、DNMT3a和DNMT3b mRNA水平，Western blotting法检测DNMTs蛋白水平。
结果
不同浓度NaAsO₂处理HaCaT细胞72 h后，HaCaT细胞ROS含量、MDA水平随染毒浓度的增加而升高（F_趋势=441.675，P < 0.001；F_趋势=22.430，P=0.012）；1.25、2.50 μmol·L^-1浓度组DNMT1 mRNA及蛋白表达水平均高于对照组（P < 0.05）。随染毒浓度的增加，DNMT1 mRNA及蛋白表达水平升高（F_趋势=30.280，P=0.001；F_趋势=40.421，P < 0.001），DNMT3a mRNA及蛋白表达水平降低（F_趋势=226.283，P < 0.001；F_趋势=30.848，P=0.001），DNMT3b蛋白表达水平升高（F_趋势=15.095，P=0.005）；各染毒组DNMT3b mRNA表达水平与对照组相比，差异无统计学意义（P > 0.05）。NAC处理后，与2.50 μmol·L-¹ NaAsO₂单独处理组相比，HaCaT细胞ROS含量、MDA水平、DNMT1 mRNA和蛋白表达水平均降低（P < 0.05），DNMT3a mRNA及蛋白水平无明显改变（P>0.05），DNMT3b蛋白水平降低（P < 0.05），mRNA水平无明显改变（P>0.05）。
结论
NaAsO₂可改变HaCaT细胞DNMTs mRNA及蛋白表达水平，这种变化可能与砷所致的氧化应激有关。

Abstract: Background
Exposure to arsenic can cause skin lesions in humans. DNA methylation changescaused by arsenic are one of its pathogenic mechanisms, but how arsenic causes DNAmethylation changes is not yet clear.
Objective
This study aims to investigate the effects of sodium arsenite (NaAsO₂) exposure onDNA methyl transferases (DNMTs) mRNA and protein expression levels of immortalized humankeratinocytes (HaCaT), and to explore the associations between oxidative stress induced byarsenic and the changes in DNMTs mRNA and protein expression levels.
Methods
HaCaT cells were treated with NaAsO₂ (0, 0.625, 1.25, and 2.50 μmol·L^-1, respectively);HaCaT cells were pretreated with antioxidant N-acetylcystein (NAC) 10.0μmol·L^-1 for 4h, and thentreated with 2.50 μmol·L^-1 NaAsO₂ for 72 h. Immunofluorescence method was used to detect thecontent of reactive oxygen species (ROS), biochemical method was used to detect the content ofmalondialdehyde (MDA), real-time fluorescence quantitative PCR was used to detect the DNMT1,DNMT3a, and DNMT3b mRNA expression levels, and Western blotting was used to detect the DNMTs protein expression levels in HaCaTcells.
Results
After 72 h of NaAsO₂ treatment, the ROS and MDA levels in HaCaT cells increased with higher dose (F_trend=441.675, P <0.001;F_trend=22.430, P=0.012); DNMT1 mRNA and protein expression levels in HaCaT cells in the 1.25 and 2.50 μmol·L^-1 groups were higherthan those in the control group (P <0.05). With the increase of NaAsO₂ dose, DNMT1 mRNA and protein expression levels in HaCaT cellsincreased (F_trend=30.280, P=0.001; F_trend=40.421, P <0.001), DNMT3a mRNA and protein expression levels decreased (F_trend=226.283, P <0.001; F_trend=30.848, P=0.001), DNMT3b protein expression level increased (F_trend=15.095, P=0.005), and there was no significant differencein the DNMT3b mRNA expression level between the NaAsO₂ treatment groups and the control group (P > 0.05). After NAC treatment, ROScontent, MDA level, and DNMT1 mRNA and protein expression levels in HaCaT cells decreased significantly compared with the 2.50 μmol·L^-1NaAsO₂ group (P <0.05); DNMT3a mRNA and protein expression levels did not change significantly (P > 0.05); DNMT3b protein expressionlevel decreased significantly (P <0.05), and its mRNA expression level did not change significantly (P> 0.05).
Conclusion
NaAsO₂ can change the mRNA and protein expression levels of DNMTs in HaCaT cells, and these changes may be related tothe oxidative stress induced by arsenic.

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