Rap1A DNA甲基化在Hcy促进巨噬细胞增殖中的作用

Effect of DNA methylation of Rap1A on macrophages proliferation induced by Hcy

目的探讨Rap1A DNA甲基化在同型半胱氨酸(Hcy)促进小鼠单核巨噬细胞RAW264.7增殖中的作用。方法将处于对数生长期的RAW264.7细胞分为对照组(Hcy 0μmol/L)和不同浓度的Hcy(20,40,60,80,100μmol/L)干预组,24 h后用XTT检测细胞活力;Edu检测细胞增殖情况;qRT-PCR和Western blot检测Rap1A的mRNA和蛋白的表达;甲基化特异性PCR法(MSP)检测Rap1A启动子区甲基化改变;激光共聚焦显微镜观察Rap1A的变化;将Rap1A干扰腺病毒转染RAW264.7细胞并用Hcy刺激,检测Rap1A的mRNA和蛋白水平的变化及细胞的增殖情况。结果与对照组相比,不同浓度的Hcy干预细胞后,细胞活力增强,其中100μmol/L Hcy浓度最为明显(P<0.01),且细胞增殖水平明显增加(P<0.01);经Hcy刺激后,细胞Rap1A的mRNA和蛋白表达量显著增加(P<0.01),启动子区甲基化水平降低(P<0.01);干扰Rap1A的表达后能够部分逆转Hcy所致的细胞增殖(P<0.01)。结论 Rap1A启动子区低甲基化介导了Hcy导致的RAW264.7细胞增殖。

Aim To investigate the role of DNA methylation of Rap1 A in Hcy-induced RAW264.7 cell proliferation. Methods RAW264.7 cells in logarithmic growth phase were divided into control group(Control) and Hcy treated groups with different concentrations(20, 40, 60, 80, 100 μmol/L Hcy). Cell viability was detected by XTT to screen optimal intervention time and concentration of Hcy after cells were treated for 24 hours. Edu test was used to analyze cell proliferation. The expression levels of Rap1 A mRNA and protein were measured by qRT-PCR and Western blot respectively. Methylation-specific PCR(MSP) was used to detect the change of Rap1 A promoter region. The changes of Rap1 A were examined by immunofluorescence staining. Rap1 A interference adenovirus was transfected into RAW264.7 cells and stimulated with Hcy to detect the changes of mRNA and protein levels of Rap1 A and cell proliferation. Results Compared with control group, the cell viability was enhanced after cells were treated with different concentration of Hcy, which was the most obvious when cells were treated by 100 μmol/L Hcy(P<0.01), as well as cell proliferation(P<0.01). But there was no time and concentration dependence. After Hcy stimulation, the expression of mRNA and protein of Rap1 A increased significantly(P<0.01). The result of MSP revealed that the methylation level of Rap1 A promoter region was decreased(P<0.01). Interfering with the expression of Rap1 A can partially reverse the proliferation of cells induced by Hcy(P<0.01). Conclusion DNA hypomethylation of Rap1 A promoter region may play an important role in macrophage proliferation induced by Hcy.