CpG methylation of the GPX3 promoter in patients with Kashin-Beck Disease potentially promotes chondrocyte apoptosis

Objective: To determine the methylation levels of CpGs in the GPX3 promoter region and explore their potential effects on the apoptosis of chondrocytes.

Methods: Blood specimens were collected from 32 participants; 16 KBD patients and 16 healthy subjects. Twenty-five CpGs in the promoter region of GPX3 were identified and detected by MALDI-TOF-MS. Methylation levels of CpGs were compared between KBD patients and healthy subjects as well as among the KBD patients with different degrees. C28/I2 human chondrocytes were treated with tBHP and Na₂SeO₃. Apoptosis in chondrocytes was examined under a fluorescence microscope.

Results: The methylation levels of GPX3-1_CpG_11 and GPX3-1_CpG_16 in KBD patients were significantly higher than those of healthy subjects (P < 0.05). The methylation levels of the other CpGs were not significantly different between the two groups (P > 0.05). The methylation level of GPX3-1_CpG_24 in KBD patients was significantly higher than those of healthy subjects (P < 0.05). MSP-PCR analysis indicated that the methylation rate of KBD group (9.41%) was significantly higher than that of healthy subjects (1.18%), and that GPX3 DNA methylation increased the risk of acquiring KBD 8 fold (OR = 8.000, 95% CI: 1.023-62.580); The mRNA expression of GPX3 in whole blood of KBD patients was lower than that of healthy subjects (P＜0.05); Compared with the control group, GPX3, GPX1 and GPX4 mRNA level of the tertbutyl hydroperoxide injury group decreased significantly (P < 0.05), after supplementation with Na₂SeO₃. The rate of chondrocytes apoptosis was decreased with the increasing of GPX3 and GPX4 mRNA levels (P＜0.05) and GPX3 mRNA showed a similar trend without statistically significant (P＞0.05).

Conclusion: The methylation patterns of CpGs in GPX3 varied in KBD patients. The experiments indicated that the increased methylation of CpGs within the GPX3 promoter may down-regulate the expression of GPX3, thereby reducing the antioxidant function of GPX3 and promoting chondrocyte apoptosis, both of which accelerates the occurrence of KBD. We therefore propose a new understanding of GPX3's potential epigenetic and genetic mechanisms that contribute to KBD.