Abstract
ObjectiveTo determine the methylation levels of CpGs in the GPX3 promoter region and explore their potential effects on the apoptosis of chondrocytes. MethodsBlood 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. ResultsThe 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). ConclusionThe 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.
Highlights
Kashin-Beck disease (KBD) is a common endemic, chronic and degenerative bone disease with the highest prevalence in China
The current study aimed to identify the divergent methylation of CpGs in GPX3 promoter regions in KBD patients, and to further analyze the effect of GPX3 promoter methylation in KBD
The results showed that the mRNA transcription levels of GPX3 in KBD patients and chondrocytes treated with tertbutyl hydroperoxide (tBHP) were all decreased significantly, the apoptosis rates of chondrocytes were decreased with logarithmic elevation of GPX3 mRNA levels
Summary
Kashin-Beck disease (KBD) is a common endemic, chronic and degenerative bone disease with the highest prevalence in China. KBD mainly affects the joints and cartilages of individuals[1, 2]. KBD is mainly distributed from the northeast to the southwest of China in a belt shaped manner, as well as parts of Eastern Siberia in Russia, and North Korea[3, 4]. The recent epidemiological survey indicated that 0.54 million individuals were suffering from KBD by the close of 2017. It is estimated that 37.2 million individuals are at high risk of developing KBD in China[5]. KBD gravely threatens the health of the population; its pathogenesis remains elusive
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