Zhang Rongqiang, Zhang Di, Yang Xiaoli, Zhang Dandan, Li Qiang, Wang Chen, Yang Xuena, Guo Hao, Xiong Yongmin
School of Public Health, Shaanxi University of Chinese Medicine, Xianyang, China; Institute of Endemic Diseases and Key Laboratory of Trace Elements and Endemic Diseases, National Health Commission of the People's Republic of China, School of Public Health, Xi'an Jiaotong University Health Science Center, No.76 Yanta West Road, Xi'an, Shaanxi 710061, China.
Institute of Endemic Diseases and Key Laboratory of Trace Elements and Endemic Diseases, National Health Commission of the People's Republic of China, School of Public Health, Xi'an Jiaotong University Health Science Center, No.76 Yanta West Road, Xi'an, Shaanxi 710061, China.
J Trace Elem Med Biol. 2022 May;71:126943. doi: 10.1016/j.jtemb.2022.126943. Epub 2022 Feb 4.
To determine the methylation levels of CpGs in the GPX3 promoter region and explore their potential effects on the apoptosis of chondrocytes.
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 NaSeO. Apoptosis in chondrocytes was examined under a fluorescence microscope.
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 NaSeO. 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).
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.
测定GPX3启动子区域CpG位点的甲基化水平，并探讨其对软骨细胞凋亡的潜在影响。
收集32名参与者的血液样本，其中16名大骨节病（KBD）患者和16名健康受试者。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）鉴定并检测GPX3启动子区域的25个CpG位点。比较KBD患者与健康受试者以及不同程度KBD患者之间CpG位点的甲基化水平。用叔丁基过氧化氢（tBHP）和亚硒酸钠（NaSeO）处理C28/I2人软骨细胞。在荧光显微镜下检测软骨细胞的凋亡情况。
KBD患者中GPX3-1_CpG_11和GPX3-1_CpG_16的甲基化水平显著高于健康受试者（P<0.05）。两组中其他CpG位点的甲基化水平无显著差异（P>0.05）。KBD患者中GPX3-1_CpG_24的甲基化水平显著高于健康受试者（P<0.05）。甲基化特异性PCR（MSP-PCR）分析表明，KBD组的甲基化率（9.41%）显著高于健康受试者（1.18%），且GPX3 DNA甲基化使患KBD的风险增加8倍（比值比=8.000，95%可信区间：1.023-62.580）；KBD患者全血中GPX3的mRNA表达低于健康受试者（P＜0.05）；与对照组相比，叔丁基过氧化氢损伤组补充亚硒酸钠后，GPX3、GPX1和GPX4的mRNA水平显著降低（P<0.05）。随着GPX3和GPX4 mRNA水平的升高，软骨细胞凋亡率降低（P＜0.05），GPX3 mRNA呈现类似趋势但无统计学意义（P＞0.05）。
KBD患者中GPX3的CpG位点甲基化模式存在差异。实验表明，GPX3启动子区域内CpG位点甲基化增加可能下调GPX3的表达，从而降低GPX3的抗氧化功能并促进软骨细胞凋亡，二者均加速了KBD的发生。因此，我们对GPX3在大骨节病发生中的潜在表观遗传和遗传机制提出了新的认识。
