2023, Vol. 27 ›› Issue (5): 714-719
Ephrin A receptor 2 DNA methylation increases in pancreatic beta cell apoptosis induced by homocysteine
Zhang Qing1, 2, 3, Gao Chunlan4, Yu Feifei1, 2, 3, Zhang Zhenghao1, 2, 3, Ma Fang1, 2, 3, Gao Yuan1, 2, 3, Li Guizhong1, 2, 3, Jiang Yideng1, 2, 3, Ma Shengchao1, 2, 3
1School of Basic Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 2Key Laboratory of Metabolic Cardiovascular Disease Research, National Health Commission of China, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3Ningxia Key Laboratory of Vascular Injury and Repair Research, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 4Yinchuan First People’s Eye Hospital, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Abstract: BACKGROUND: Increased homocysteine levels lead to apoptosis of pancreatic β cells, 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 pancreatic β cells.
METHODS: Mouse insulinoma cell lines (Min6) were cultured in vitro and divided into control group (0 µmol/L homocysteine) and homocysteine group (120 µmol/L homocysteine). After 48 hours of intervention in the cells, immunofluorescence and western blot were used to test the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3 in pancreatic islet β cells 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 pancreatic β cells 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 pancreatic β cells were significantly higher in the homocysteine group (P < 0.05). To conclude, EphA2 DNA hypermethylation plays a significant role in homocysteine-induced pancreatic β cell apoptosis and DNMT1 may be involved in its hypermethylation process.
Key words: pancreatic islet β cells, homocysteine, apoptosis, Ephrin A receptor 2, DNA methylation