赖氨酸甲基转移酶PR-Set7在亚砷酸钠致人永生化皮肤角质形成细胞DNA损伤中的作用

Effects of lysine methyl transferase PR-Set7 in DNA damage induced by sodium arsenite in human immortalized skin keratinocytes

目的探讨组蛋白甲基转移酶PR-Set7在亚砷酸钠致人永生化皮肤角质形成细胞(Ha Ca T细胞)DNA损伤中的作用机制。方法将处于对数生长期的Ha Ca T细胞暴露于含终浓度分别为0(对照)、1.25、2.5、5、10μmol/L亚砷酸钠的DMEM高糖培养基染毒24 h。采用MTT法测定Ha Ca T细胞的存活情况。另设对照(24 h)组、亚砷酸钠(10μmol/L,24 h)染毒组、PR-Set7小干扰RNA干扰组(Si PR-Set7,50 nmol/L,48 h)和联合暴露(50 nmol/L Si PR-Set7干扰48 h后,10μmol/L亚砷酸钠暴露24 h)组。分别采用q RT-PCR法检测PR-Set7 m RNA的表达水平,采用Western blot法检测H4K20me1和PR-Set7蛋白的表达水平,采用单细胞凝胶电泳法检测DNA损伤水平。结果与对照组相比,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞的存活率均较低,差异有统计学意义(P<0.05)。与对照组比较,5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均升高,而H4K20me1蛋白的表达水平降低;2.5、5、10μmol/L亚砷酸钠染毒组Ha Ca T细胞尾部DNA%及尾矩升高,差异有统计学意义(P<0.05)。随着亚砷酸钠染毒浓度的升高,Ha Ca T细胞PR-Set7蛋白和m RNA的表达水平均呈上升趋势,而H4K20me1蛋白和m RNA的表达水平呈下降趋势,DNA损伤水平(尾部DNA%及尾矩)呈上升趋势。与对照组比较,Si PR-Set7干扰组Ha Ca T细胞PR-Set7蛋白的表达大幅下降;亚砷酸钠染毒组、Si PRSet7干扰组和联合暴露组Ha Ca T细胞H4K20me1蛋白的表达水平均降低,而尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。与亚砷酸钠染毒组比较,联合暴露组Ha Ca T细胞PR-Set7蛋白和m RNA以及H4K20me1蛋白的表达均降低,而Ha Ca T细胞尾部DNA%及尾矩均升高,差异有统计学意义(P<0.05)。结论在对Ha Ca T细胞进行PR-Set7干扰后,亚砷酸钠可能通过抑制组蛋白甲基转移酶PR-Set7蛋白及m RNA的表达水平,从而降低H4K20me1的修饰水平,影响Ha Ca T细胞DNA损伤修复能力,使DNA损伤加重。

Objective To investigate the mechanism of histone methyl transferase PR-Set7 in DNA damage of human immortalized keratinocytes（Ha Ca T cells） induced by sodium arsenite. Methods The Ha Ca T cells in logarithmic growth period were exposed to DMEM high glucose medium which containing sodium arsenite with final concentrations of 0（control）,1.25,2.5, 5 and 10 μmol/L respectively for 24 h. MTT method was used to detect the survival of Ha Ca T cells in each group. The control group（24 h）, sodium arsenite exposure group（10 μmol/L, 24 h）, small interfering RNA of PR-Set7 group（si PR-Set7,50 nmol/L, 48 h） and the combined exposure group（50 nmol/L si PR-Set7 interference 48 h, 10 μmol/L sodium arsenite exposure 24 h） were designed. The expression levels of PR-Set7 m RNA were measured by q RT-PCR. The protein expression levels of H4 K20 me1 and PR-Set7 were detected by Western blot. Single cell gel electrophoresis was used to observe the damage degree of DNA（tail DNA% and tail moment） in Ha Ca T cells. Results Compared with the control group,the expression levels of PR-Set7 protein and m RNA in 5 and 10 μmol/L sodium arsenite group increased,while the expression level of H4 K20 me1 protein decreased,the tail DNA% and Tail moment of Ha Ca T cells increased in 2.5,5 and 10 μmol/L sodium arsenite group（P〈0.05）, with a dose dependent manner. Compared with the control group,the expression of PR-Set7 protein and m RNA in small interfering RNA of PR-Set7 group decreased,Ha Ca T cells in the sodium arsenite group,small interfering RNA of PR-Set7 group and combined exposure group,the expression level of H4 K20 me1 protein decreased,while the tail DNA%and tail moment increased（P〈0.05）. Compared with sodium arsenite group,the expression of PR-Set7 protein and m RNA and H4 K20 me1 protein expression in combined exposure group decreased,while tail DNA% and tail moment were both increased（P〈0.05）. Conclusion After the low expression of PR-Set7 in Ha Ca T cells,sodium arsenite may reduce the level of H4 K20 me1 by inhibiting the transcription and protein expression of histone methyltransferase PR-Set7 m RNA,affecting the ability of DNA damage repairment and aggravating DNA damage.