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, including 16 KBD patients and 16 healthy subjects. Twenty-ve CpGs in the promoter region of GPX3 were identied 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 Na 2 SeO 3 . Apoptosis in chondrocytes was examined under a uorescence microscope. Results: The methylation levels of GPX3-8_CpG_11, GPX3-8_CpG_16, GPX3-8_CpG_20 in KBD patients were signicantly higher than those of healthy subjects (P <0.05). The methylation levels of the other CpGs were not signicantly different between the two groups (P> 0.05). The methylation level of GPX3-8_CpG_24 in KBD patients with degree III was signicantly higher than those in KBD patients with degree I/II (P <0.05) MSP-PCR analysis indicated that the methylation rate of KBD group (9.41%) was signicantly 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 (cid:0) 0.05); Compared with the control group, GPX3, GPX1 and GPX4 mRNA level of the tertbutyl hydroperoxide injury group decreased signicantly (P <0.05), after supplementation with Na 2 SeO 3 . The logarithmic increase in mRNA levels of GPX3, GPX1 and GPX4 decreased the rate of apoptosis in chondrocytes. 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 development of KBD. The results showed that the mRNA transcription levels of GPX3 in KBD patients and chondrocytes treated with tBHP were all decreased signicantly, similarly, the apoptosis rates of chondrocytes were decreased with logarithmic elevation of GPX3 mRNA levels. The results indicated that increased levels of methylation in GPX3 CpGs may down-regulate the GPX3 mRNA transcription levels, thereby reducing its antioxidant function, promoting apoptosis of chondrocytes and consequently accelerating the development of KBD. CpG methylation levels and their potential effect on apoptosis of chondrocytes. The results indicated that increased levels of GPX3 CpGs methylation may down-regulate GPX3 mRNA transcription and induce chondrocyte apoptosis. Our study provided valuable clues for exploring the potential interaction between the CpG methylation (epigenetics) and mRNA expression (genetics) of GPX3 in KBD. Integrated analysis of the genetic and epigenetics information of genes will unravel the complexity of chronic bone diseases, such as KBD[27, 28].


Background
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].
Geographically, 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. Currently, 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; however, its pathogenesis remains elusive.
However, the roles of selenium in the occurrence and development of KBD has not been ascertained. Selenium usually performs its biological roles in the human body as selenoprotein, of which its physiological function still needs to be systematically studied. The antioxidant effect of selenoprotein is mainly attributed to the oxidation state of the selenium atom. Such antioxidant enzymes prevents DNA damage caused by oxidative stress, which is related to upregulated apoptosis and necrosis of KBD articular chondrocytes [9][10][11]. The above evidence indicates that the antioxidant properties of selenoproteins can protect chondrocytes from apoptosis and necrosis.
In recent years, studies have con rmed that, despite low environmental selenium concentrations, the individual's genetic characteristics also play an important role in the occurrence and development of KBD [12]. Therefore, the genetic factors contributing to KBD have received increasing attention.
Consequently, a number of KBD susceptible genes were identi ed from some studies through modern molecular biology research technologies [13][14][15]. However, studies on the genetic factors contributing to KBD can only explain its pathogenesis partly. Researchers are rapidly discovering that epigenetic elements may also affect gene function by regulating gene expression levels [16], hence exhibiting a series of effects on human physiology.
Glutathione peroxidase family are key members of selenoproteins that catalyze the reduction of organic hydroperoxides and hydrogen peroxide (H 2 O 2 ) by glutathione, and thereby inhibiting oxidative cell damage [17,18]. Experiments have showed that downregulation of GPX3 expression by hypermethylation of its promoter, has been observed in a wide spectrum of human malignancies such as Osteoarthritis (OA) and KBD [16]. Therefore, genetic and epigenetic interactions of GPX3 may affect human health. However, methylation levels of the GPX3 promoter has not been quanti ed in KBD.
In our present study, CpGs of GPX3 in the promoter region were identi ed and determined. Matrix assisted laser desorption/ionization time of ight mass spectrometry (MALDI-TOF-MS) was employed to quantitate the methylation of CpGs explored their potential effect on the apoptosis of chondrocytes. Through these evaluations, 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.

Study population
The study population was selected from KBD endemic areas of Shaanxi province, China. A biological specimen library was created from blood samples drawn from 226 KBD patients and 248 healthy subjects (Table 1). All the study participants received prudent radiographic examination. The national diagnosis criteria of KBD (WS/T207-2010) was used to select patients with KBD. Individuals with genetic bone and cartilage diseases, arthritis related disease and other skeletal disorders were excluded from this study. Individuals who met the selection criteria were recruited to participate in the study after submitting their signed informed consent. The proposed study design met the approval of the Human Ethics Committee of Xi'an Jiaotong University, People's Republic of China.

Quantifying the mRNA expression in KBD patients and chondrocytes
Total RNA from KBD patients and controls (n=8 in each group), as well as chondrocytes (n=3 in each group), was extracted using the Trizol KIT (Life Technologies, Carlsbad, CA). RNA extracts were reversetranscribed into cDNA using the RevertAidTM First Strand cDNA Synthesis Kit (MBI, Fermentas, Vilnius, Lithuania) following the manufacturer's instructions. Relative quanti cation of GPX3, GPX1 and GPX4 mRNA was performed by iQe5 quantitative real-time PCR Detection Systems (qRT-PCR) (Bio-Rad, Philadelphia, PA) with β-actin as a reference. The sequences of primers are listed in Table 2. qRT-PCR was performed in a 20 μL reaction mixture containing cDNA (1.6 μL), each primer(0.8μL), 2×SYBR Premix Ex Taq (10μL) (Takara, Mountain View, CA), and ddH 2 O (6.8 μL) using the TaqMan method (94°C for 2 min, and 40 cycles of 94°C for 10 s and 72°C for 30 s). All reactions were performed in duplicate. Relative expression levels of GPX3, GPX1 and GPX4 mRNAs were normalized to β-actin and analyzed by iQe5 software (version 2.0, Bio-Rad, Philadelphia, PA). which are also illustrated in Figure 1A. The target fragment of GPX3 contained 25 CpGs, which is showed in Figure 1B Next, a 50μL PCR mixture containing 5μL 10×PCR buffer, 4μL dNTP mixture, 1μL of each primer, 3μL bisul te-modi ed DNA, 35.75μL ddH2O, and 0.25μL hot-start Taq-polymerase (Takara, Mountain View, CA) for each blood sample was prepared. PCR conditions were set as: 94 °C for 10 min (initial denaturation), followed by 40 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and a nal extension of 72 °C for 10 min. Finally, PCR products were resolved in 2% agarose gels, stained with ethidium bromide, and visualized in ultraviolet light (GBox F3, Syngene, UK).

Statistical analysis
Quantitative data was presented as mean ± standard deviation (SD). Groups were compared using the student's t-test. Curve-tting method was used for obtaining the correlation between GPX1, GPX3 and GPX4 mRNA expressions and apoptosis rate of chondrocytes. All statistical analyses were performed using SPSS 23.0, and the signi cance level α=0.05 were considered to have statistical signi cance (SPSS Inc., Chicago, IL, USA).

Baseline characteristics of study population
226 KBD patients and 248 healthy controls with matched frequency of age and gender were recruited in this study. No statistically signi cant differences were seen between the cases and controls in age (t=1.305, P=0.193) and sex (x 2 =1.914, P=0.167). The demography of the two groups are shown in Table  1. 2.2 The CpGs methylation of GPX3 of KBD randomly from the study population to detect CpG methylation detection (Table 3). Figure 2 illustrates the methylation pro les of 14 CpGs (or CpG Units) in the promoter region of GPX3. The methylation levels of GPX3-8_CpG_11, GPX3-8_CpG_16, GPX3-8_CpG_20 in KBD patients were signi cantly higher than those in the healthy subjects (P <0.05). The methylation levels of the other CpGs were not signi cantly different between the two groups (P > 0.05) (Figure 3).

The CpGs methylation of GPX3 in KBD patients with different degrees
The methylation levels of 14 CpGs (or CpG Units) in the promoter region of GPX3 between KBD patients with degree I/II and III are shown in Figure 4. The methylation level of GPX3-8_CpG_24 in KBD patients with degree III was signi cantly higher than those in KBD patients with degree I/II (P <0.05).

Validation by MSP-PCR
MSP-PCR was used to detect the methylation status of GPX3promotor region in KBD patients and healthy subjects (n=85/group) quantitatively ( Table 4). The amplicons were resolved by 2% agarose gel electrophoresis with band patterns showing that 8 (9.41%) patients with KBD and 1 (1.18%) healthy controls developed methylation in GPX3. After x 2 test, the difference of methylation rates of GPX3 between the two groups was signi cant (x 2 = 4.396, P = 0.036), indicating that the methylation rate of KBD group was signi cantly higher than that of control group. Also, GPX3 DNA methylation increased the risk of KBD 8-fold (OR = 8.000, 95% CI: 1.023-62.580) ( Table 5). The results of MSP-PCR and MALDI-TOF-MS were harmonious.

2.5The mRNA transcription level of GPX3 in KBD patients
GPX3 mRNA level in whole blood was detected by qRT-PCR with β-actin as a quantitative control. Results showed the mRNA expression of GPX3 in whole blood of KBD patients was lower than that of healthy subjects (P 0.05) ( Figure 5).

The effect of Oxidative damage and Selenium on chondrocytes apoptosis
The results showed that the apoptosis rate in group C, O, S2, OS1, OS2, OS3 was (1.30 ± 0.32)%, (65.96 ± 3.98)%, (2.03 ± 0.36)%, (14.54 ± 2.02)%, (4.15 ± 0.19)%, and (14.74 ± 2.56)%, respectively. The apoptosis rates of the six groups was signi cantly different from each other (F = 595.157, P <0.001). Pairwise comparison using LSD analysis suggested that the apoptosis rate in group O was signi cantly higher than that in group C (P <0.001) while apoptosis rates of group OS1, OS2, OS3 were signi cantly lower than that of group O (P <0.001). These results indicate that tBHP-induced oxidative damage can increase apoptosis of chondrocytes, while in contrast, supplementation with Na 2 SeO 3 can decrease apoptosis of chondrocytes.

The mRNA transcription levels of GPX3 in chondrocytes
The changes of GPX3 mRNA levels in chondrocytes treated with tBHP and Na 2 SeO 3 was further examined. The results showed that GPX3 mRNA level of group O decreased signi cantly compared with group C (P <0.05). However, GPX3 mRNA levels were elevated upon supplementation with Na 2 SeO 3 ( Figure 6A).

The mRNA transcription levels of GPX1 and GPX4 in chondrocytes
The changes in mRNA levels of GPX1 and GPX4, the two main members of GPXs family, in chondrocytes treated with tBHP and Na 2 SeO 3 was also examined to observe whether they exhibited similar trends in mRNA transcription. The results showed that GPX1 and GPX4 mRNA levels both decreased signi cantly in group O in comparison with group C (P <0.05). However, GPX1 and GPX4 mRNA levels were increased after supplementation with Na 2 SeO 3 ( Figure 6B-C).

The effect of GPX1, GPX3 and GPX4 mRNA transcription levels on chondrocytes apoptosis
In order to understand the effect of GPX1, GPX3 and GPX4 mRNA levels on chondrocytes apoptosis, curve tting was used to correlate GPX1, GPX3, GPX4 mRNA levels with apoptosis rate of chondrocytes. The results showed that the rate of apoptosis was decreased with the elevation of GPX1, GPX3, GPX4 mRNA levels. In addition, the increasing trends of GPX1, GPX3, GPX4 mRNA levels exhibited similar "L" curves ( Figure 7).

Discussion
We aimed to evaluate the CpG methylation level of GPX3 of KBD quantitatively. Primers targeting the 25 CpGs were therefore designed to test the CpG methylation levels by MALDI-TOF-MS. Signi cant variation in methylation of GPX3 CpGs in KBD blood were identi ed. To substantiate the pathogenetic implication for KBD, C28/I2 human chondrocytes treated with tBHP were cultured to explore the potential effect of methylation patterns in the CpGs of GPX3 on KBD development. The results showed that the mRNA transcription levels of GPX3 in KBD patients and chondrocytes treated with tBHP were all decreased signi cantly, similarly, the apoptosis rates of chondrocytes were decreased with logarithmic elevation of GPX3 mRNA levels. The results indicated that increased levels of methylation in GPX3 CpGs may downregulate the GPX3 mRNA transcription levels, thereby reducing its antioxidant function, promoting apoptosis of chondrocytes and consequently accelerating the development of KBD.
Classic chondrocytic necrosis in articular cartilage, excessive apoptosis, dedifferentiation of chondrocytes, and chronic in ammation are the main pathological features of KBD, which are widely accepted. Findings about the bene cial effects of various antioxidants on KBD are gradually increasing.
Selenium, assimilated in humans as selenocysteine (Sec) in selenoproteins such as glutathione peroxidases(GPXs), participates in antioxidant defense in human bodies [19]. More than 50 selenoproteins have currently been identi ed and 25 of them can be found in humans [19,20]. Nevertheless, many of their structure and speci c biological functions remains an enigma. Glutathione peroxidases (GPXs) are a big family of antioxidant enzymes which can actively reduce H 2 O 2 and organic hydroperoxides to inhibit the ROS-induced damage to cell membranes and proteins [21]. Possibly, GPXs protect cells from oxidative damage. In mammals, eight glutathione peroxidases (GPX1-GPX8) have been identi ed so far, of which GPX1-GPX4 and GPX6 are selenium-containing GPXs [22].
Currently and collectively, the biological functions of GPX1, GPX3, and GPX4 are widely and mostly studied. GPX1 is activated during cartilage formation and is responsible for eliminating reactive oxygen species (ROS), which are essential for chondrogenic induction [23]. Results reported here showed that GPX1 knockdown can impair gene expression of Col II and aggrecan and also suppress the proliferation of chondrocytes. Therefore, GPX1 is necessary for chondrogenic induction in ATDC5 cells [24]. Another study showed that GPX3 was signi cantly increased in bone samples from hip fracture patients, which suggested increased antioxidative activity in the samples [25]. The latest evidence showed that the methylation and expression of GPX3 vary in KBD blood samples [16], which may have an important role in this bone disease occurrence and development. Furthermore, the mRNA level of GPX4 was strikingly decreased in the blood of KBD patients and the polymorphisms of two SNPs (rs713041, rs4807542) of GPX4 may be related to the development of KBD. On that account, GPX4 can serve as a potential susceptibility biomarker for KBD [26]. The above evidence strongly suggests that these seleniumcontaining GPXs may play essential roles in the occurrence of KBD, which is worth exploring further.
In addition to the genetic factors of GPXs, their epigenetic studies are also an important hotspot for study. However, quantitative research on the methylation levels of GPXs in KBD have not been reported. In our present study, we paid special attention to the quantitative assessment of GPX3 CpG methylation levels and their potential effect on apoptosis of chondrocytes. The results indicated that increased levels of GPX3 CpGs methylation may down-regulate GPX3 mRNA transcription and induce chondrocyte apoptosis. Our study provided valuable clues for exploring the potential interaction between the CpG methylation (epigenetics) and mRNA expression (genetics) of GPX3 in KBD. Integrated analysis of the genetic and epigenetics information of genes will unravel the complexity of chronic bone diseases, such as KBD [27,28].
It has been recognized that epigenetic deregulation through genetic and environmental elements could result in delayed or blocked cancer development [29]. The genetic pro le of an individual could provide the underlying susceptibility factors and triggers for the evolution of a complex disease (cancer, bone disease, etc.). Epigenetic regulation also has very important implications for development of such diseases [30]. Increasing evidence showed that unraveling the interplay between genetic and epigenetic parameters becomes increasingly essential for interpreting the etiology of complex diseases [27,28,30]. The ndings of our present study provide preliminary clues and new evidence that could illustrate the interplay between genetic and epigenetic parameters of GPX3 in the onset of KBD.

Conclusions
The methylation pro les of CpGs in the promoter region of GPX3 in the whole blood of KBD patients were detected and their potential effect on the apoptosis of chondrocytes was explored. It was initially suggested that there may be an interplay between the CpGs methylation in the promoter region of GPX3 and GPX3 expression in KBD patients, which stimulated chondrocyte apoptosis. Our present study provides a new perspective and new clue for mastering the pathogenic process of KBD.

Declarations
Ethics approval and consent to participate Individuals were recruited to participate in the study after submitting their signed informed consent. The proposed study design met the approval of the Human Ethics Committee of Xi'an Jiaotong University, People's Republic of China.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analyzed during this study are included in this published article.    Figure 1 The information of CpG methylation sites in primer GPX3 #8 and the sequence of target fragment in the promoter region of GPX3 (A: The dots represent the CpG sites to be tested in the target fragment; B: The blue bases represent CpG sites to be tested).