H3K9me2调控核苷酸切除修复相关基因转录在亚砷酸钠致L-02细胞DNA损伤中的作用

H3K9me2 regulates the transcription of nucleotide excision repair related genes in DNA damage of normal hepatocytes induced by sodium arsenic

目的了解不同剂量亚砷酸钠(NaAsO2)对正常人肝细胞(L-02细胞)核苷酸切除修复(NER)相关基因mRNA转录水平及其启动子区组蛋白H3第9位赖氨酸二甲基化(H3K9me2)修饰水平的影响。方法以0(对照)、5、10、20μmol/L的NaAsO2处理L-02细胞24 h(n=3),采用单细胞凝胶电泳法(SCGE)检测L-02细胞DNA损伤情况[Olive尾距(OTM)、尾部DNA百分含量(Tail DNA%)];实时荧光定量PCR法检测NER相关基因着色性干皮病(XP)基因A(XPA)、XP基因D(XPD)、XP基因F(XPF)mRNA表达水平;定量染色质免疫沉淀(CHIP)技术检测XPA、XPD、XPF启动子区(CHIP1、CHIP2)H3K9me2修饰水平。结果OTM、Tail DNA%水平与染砷剂量均呈正相关(对照和5、10、20μmol/L染砷组分别为0.35±0.09、0.56±0.18、3.18±0.31、4.52±0.55,0.72±0.05、1.34±0.26、3.93±0.43、5.47±0.65,r=0.927、0.948,P均<0.05)。与对照组比较,10、20μmol/L染砷组L-02细胞XPA、XPD、XPF mRNA表达水平均较低(P均<0.05)。与对照组比较,20μmol/L染砷组L-02细胞XPA、XPD、XPF启动子区(CHIP1、CHIP2)H3K9me2富集水平均较高(P均<0.05)。结论砷通过增加NER相关基因启动子区(CHIP1、CHIP2)H3K9me2富集水平,抑制NER相关基因转录,进而降低L-02细胞DNA损伤修复能力,导致DNA损伤加重。

Objective To investigate the effects of different doses of sodium arsenic(NaAsO2)on mRNA transcription levels of nucleotide excision repair(NER)related genes in normal hepatocytes(L-02 cells)and the modification levels of histone H3 ninth lysine dimethylization(H3K9me2)in the promoter region.Methods L-02 cells were treated with 0(the control group),5,10 and 20μmol/L NaAsO2 for 24 h(n=3).Single cell gel electrophoresis(SCGE)was used to detect DNA damage[Olive tail distance(OTM)and Tail DNA percentage(Tail DNA%)]in L-02 cells.The mRNA expression levels of Xeroderma pigmentosum(XP)gene A(XPA),XP gene D(XPD)and XP gene F(XPF)were detected by real-time fluorescence quantitative PCR.The modification levels of H3K9me2 in XPA,XPD and XPF gene promoter regions(CHIP1 and CHIP2)were detected by quantitative chromatin immunoprecipitation.Results OTM and Tail DNA%were positively correlated with arsenic doses(in the control and 5,10 and 20μmol/L arsenic exposure groups,the values were 0.35±0.09,0.56±0.18,3.18±0.31,4.52±0.55,0.72±0.05,1.34±0.26,3.93±0.43,5.47±0.65,respectively,r=0.927,0.948,P<0.05).Compared with the control group,the mRNA expression levels of XPA,XPD and XPF in L-02 cells of 10 and 20μmol/L arsenic exposure groups were significantly lower(P<0.05).Compared with the control group,the enrichment levels of H3K9me2 in XPA,XPD and XPF gene promoter regions(CHIP1 and CHIP2)in L-02 cells of 20μmol/L arsenic exposure group were significantly higher(P<0.05).Conclusion Arsenic may inhibit the transcription of NER related genes by increasing the enrichment level of H3K9me2 in the promoter regions(CHIP1 and CHIP2)of NER related genes,thereby reduce the DNA damage repair ability of L-02 cells,resulting in the aggravation of DNA damage.