同型半胱氨酸致胰岛β细胞凋亡中肝配蛋白A型受体2 DNA甲基化升高

Ephrin A receptor 2 DNA methylation increases in pancreatic beta cell apoptosis induced by homocysteine

背景:同型半胱氨酸水平增加会导致胰岛β细胞发生凋亡,但其具体机制尚不明确。目的:探讨胰岛β细胞中肝配蛋白A型受体2及其启动子区DNA高甲基化的具体机制。方法:体外培养小鼠胰岛β细胞株Min6,将其分为对照组(0μmol/L同型半胱氨酸)和同型半胱氨酸组(120μmol/L同型半胱氨酸)。干预细胞48 h后,采用免疫荧光和Western blot法检测2组细胞中凋亡相关蛋白Bax、Bcl-2、半胱氨酰天冬氨酸特异性蛋白酶3表达情况;Western blot法检测DNA甲基化相关蛋白DNMT1、DNMT3a的表达水平;实时荧光定量PCR检测两组细胞中肝配蛋白A型受体2 mRNA水平;Western blot检测肝配蛋白A型受体2的蛋白表达情况;巢式甲基化特异性PCR检测EphA2启动子区DNA甲基化水平。结果与结论:①与对照组相比,同型半胱氨酸组胰岛β细胞中凋亡相关蛋白Bax和半胱氨酰天冬氨酸特异性蛋白酶3表达明显升高,Bcl-2表达明显下降;肝配蛋白A型受体2的mRNA和蛋白表达水平明显下降(P<0.05);②与对照组相比,同型半胱氨酸组肝配蛋白A型受体2 DNA甲基化水平明显升高(P<0.05),同型半胱氨酸组胰岛β细胞中DNMT1蛋白表达明显增高(P<0.05);③提示肝配蛋白A型受体2 DNA高甲基化在同型半胱氨酸致胰岛β细胞凋亡中的作用明显,而DNMT1可能参与其高甲基化过程。

BACKGROUND:Increased homocysteine levels lead to apoptosis of pancreaticβcells,but the exact mechanism remains unclear.OBJECTIVE:To explore the specific mechanism of DNA hypermethylation of Ephrin A receptor 2(EphA2)and its promoter region in pancreaticβcells.METHODS:Mouse insulinoma cell lines(Min6)were cultured in vitro and divided into control group(0μmol/L homocysteine)and homocysteine group(120μmol/L homocysteine).After 48 hours of intervention in the cells,immunofluorescence and western blot were used to test the expression of apoptosisrelated proteins Bax,Bcl-2,and Caspase-3 in pancreatic isletβcells of the two groups.The expression levels of DNA methylation-related proteins DNMT1 and DNMT3a were detected by western blot.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the level of EphA2 mRNA.Western blot was used to detect the protein expression of EphA2.Nested methylation-specific PCR was used to detect the level of DNA methylation in the promoter region of EphA2.RESULTS AND CONCLUSION:Compared with the control group,the expression of apoptosis-related proteins Bax and Caspase-3 in the pancreaticβcells was significantly increased in the homocysteine group,and the expression of Bcl-2 was significantly decreased;the mRNA and protein expression levels of EphA2 were significantly decreased(P<0.05).Compared with the control group,the EphA2 DNA methylation level and the expression of DNMT1 protein in the pancreaticβcells were significantly higher in the homocysteine group(P<0.05).To conclude,EphA2 DNA hypermethylation plays a significant role in homocysteine-induced pancreaticβcell apoptosis and DNMT1 may be involved in its hypermethylation process.