丝/苏氨酸蛋白激酶DNA甲基化在同型半胱氨酸致血管内皮细胞凋亡中的作用

Effect of mammalian sterile 20-like kinase 1 DNA methylation in apoptosis of homocysteine-induced vascular endothelial cells

目的探讨丝/苏氨酸蛋白激酶(MST1)DNA甲基化在同型半胱氨酸(Hcy)致血管内皮细胞凋亡中的作用。方法培养人脐静脉血管内皮细胞(HUVECs),用100μmol/L Hcy干预48 h。MTT法检测HUVECs的存活率;Hoechst染色观察HUVECs的凋亡情况;Western blot检测MST1、Bcl-2、BAX和DNMT1的蛋白表达;巢式降落式PCR(nMS-PCR)法检测MST1启动子区DNA甲基化改变。结果与正常对照组相比,Hcy处理后HUVECs的存活率明显降低(P <0.01);Hoechst 33258染色后Hcy组可见核固缩、凋亡小体等典型的凋亡形态学特征;Western blot检测BAX的蛋白表达增加了1.23倍,而Bcl-2的蛋白表达及Bcl-2/BAX明显下调(P <0.05);MST1的蛋白表达升高(P <0.01),与Bcl-2/BAX呈负相关(r^2=0.844 6,P <0.001);同时,MST1 DNA甲基化水平及DNMT1蛋白表达下降(P <0.01)。结论 MST1表达增高可能在Hcy致HUVECs凋亡中发挥重要作用,而DNMT1表达下调引起的MST1启动子区DNA低甲基化可能是其表达增高的重要机制。

Objective To investigate the role of MST1 DNA methylation in the apoptosis of vascular endothelial cells induced by Hcy. Methods Human umbilical vein endothelial cells( HUVECs) were cultured with 100 μmol/L Hcy for 48 h. The survival rate of HUVECs was detected by MTT assay. The apoptosis of HUVECs was measured by Hoechst staining. The protein expression levels of MST1,Bcl-2,BAX and DNMT1 were assessed by Western blot. Nested methylation specific polymerase chain reaction( nMS-PCR) was used for analysis of the meth ylation pattern in promoter regions of MST1 gene. Results Compared with the control group,the survival rate of endothelial cells was significantly decreased(P<0.01). With Hoechst 33258 staining,the nucleus in Hcy group showed an appearance of typical morphological features of apoptosis,such as karyopyknosis,apoptotic body,etc. Western blot assay showed the protein expression of BAX increased by 1.23 times, while the Bcl 2 protein expression and the ratio of Bcl-2/BAX were significantlydecreased(P<0.05). Additionally,the protein expression of MST1 was significantly increased(P<0.01), which was negatively correlated with the ratio of Bcl 2/BAX(r^2 = 0.844 6, P<0.001). At the same time,both DNA methylation level in MST1 promoter region and DNMT1 expression were significantly decreased(P<0.01). Conclusion The up regulation of MST1 plays a significant role in the apoptosis of endothelial cells induced by Hcy,which may be attributed to the hypomethylation of MST1 promoter region medi ated by the suppression of DNMT1 expression level.