DNA methylation is an extremely abundant chemical modification. Its abnormal changes (including hypermethylation or hypomethylation) are related to gene expression and contribute to the development of diseases. At present, most of the research on DNA methylation has focused on CpG islands or short DNA fragments that are essential for transcriptional regulation and rich in high CpG sites, such as gene promoter regions [18, 19]. When these CpG sites that specifically bind to transcription factors are hypermethylated, the combination of promoters and transcription factors is reduced, thereby silencing or inhibiting gene transcription and expression. Many studies on the methylation of target genes have clarified the relationship between abnormal methylation and disease [20, 21]. In recent years, increasing evidence has shown that DNA methylation modification also affects the pathogenesis of nontraumatic ONFH. Some related methylation genes and loci have also been reported, such as the ABCB1 gene, RUNX2 gene, FZD1 gene, and CARS gene.
In previous studies, the methylation status of the OPG/RANK/RANKL system was closely related to bone metabolic diseases. For example, Delgado-Calle, J. et al. [22] studied the role of DNA methylation in regulating the OPG/RANKL system in human bone. The results showed that in HEK-293 and HOS-TE85 cells, the degree of methylation in the CPG-enriched region of the OPG gene was higher, and its expression was reduced. RANKL was hypermethylated in the osteoblast cell line HOS-TE85 and renal-derived HEK-293 cells, and its mRNA expression was decreased. This suggests that DNA methylation inhibits the expression of OPG and RANKL genes. Wang Peng et al. [23] found that in the bone tissue of osteoporotic fractures, the CpG methylation status of the RANKL gene promoter was remarkably reduced, and the expression was significantly increased. However, for the OPG gene, a higher degree of methylation and a lower expression level were found. It has not been reported whether methylation changes in this pathway also contribute to the occurrence of steroid-related ONFH. Therefore, we investigated the methylation status of CpG islands in the OPG, RANK, and RANKL genes in patients with steroid-related ONFH.
In the case group of this study, we found that the methylation levels of 4 CpG units in the OPG gene, 15 CpG units in the RANK gene, and 10 CpG units in the RANKL gene were significantly higher than those in the healthy control group (p < 0.05). In the control group, the methylation rates of 3 CpG units in the OPG gene, 10 CpG units in the RANK gene, and 6 CpG units in the RANKL gene were lower than those in the case group (p < 0.05). Obviously, the degree of methylation of the OPG, RANK, and RANKL genes in patients with steroid-induced ONFH is different from that of healthy people. Hypermethylation is more significant in the pathogenesis of the disease. In addition, we found that two hypermethylated CpG units in the OPG gene, twelve hypermethylated CpG units in the RANK gene, and ten hypermethylated CpG units in the RANKL gene increased the risk of steroid-related ONFH (p < 0.05). To avoid the possible influence of age (the levels of methylation may be higher with age [24]) and sex on methylation, the two groups in this study were matched. The logistic regression analysis has also been revised. Because the OPG/RANK/RANKL pathway is also very important for the epigenetic regulation of obesity [25], this may also be one of the reasons for the difference in high- and low-density proteins between the two groups. Because of the small sample size, we did not conduct further stratified analysis on the course of the disease or physical indicators (such as age, sex, and BMI), which would reduce the efficiency of the test.
In other steroid diseases, methylation of the OPG, RANK, and RANKL genes has not been reported. However, the possible effect of steroids on gene methylation needs to be considered when designing case and control groups. All patients receiving systemic steroid medication are at risk of steroid-induced ONFH. Among the types of patients, SLE with ONFH is more common (approximately 30%), followed by inflammatory diseases (such as pneumonia, nephritis, hepatitis), blood system diseases (for example, thrombocytopenic purpura, aplastic anaemia), connective tissue diseases, and other diseases (for instance, eye diseases and skin diseases) [26]. Most of the patients included in this study were treated with steroids due to inflammatory disease, which resulted in ONFH. There were no healthy people receiving steroid treatment. The control group included healthy subjects who were widely used in most of the studies, rather than patients receiving steroid treatment with other conditions [27]. The reason is that even if the patients with SLE and ONFH, which accounted for the highest proportion of the disease, were taken as the control group, the resulting statistical conclusion could only prove the difference between the two diseases. At present, some studies have set up multiple control groups, such as the healthy group, SONFH group, and SLE group at the same time [28], hoping to reduce the interference of hormones on disease. However, whether this approach can completely counteract the effects of hormones on disease remains in question. In addition, the association between the degree of DNA methylation and hormone levels has not been established, mainly because of the lack of accurate statistics on hormone intake in the case group.
The regulatory effects of OPG, RANK and RANKL on osteoclasts are self-evident. The hypermethylation of CpG islands in the OPG, RANK, and RANKL genes may reduce the expression of related mRNAs, thereby downregulating the OPG/RANK/RANKL signalling pathway and leading to osteoclast dysfunction in patients with steroid-related ONFH. As a result, osteoclasts in the bone marrow of the femoral head increase, bone resorption decreases, and the structure of bone trabeculae becomes thin and disordered. Bone density decreases, leading to severe osteoporosis. The femoral head is prone to compression fractures, resulting in collapse and necrosis. Changes in the methylation status of this pathway may also lead to disorders of endothelial cell metabolism and function. On the one hand, the damaged microvascular endothelial cells can form micro-thromboses and block local blood vessels, leading to ischaemia and necrosis of the femoral head in the innervated area. On the other hand, a large number of reactive oxygen species are produced, which reduces the synthesis of vasodilatory substances and further aggravates bone cell damage. This intensifies the progression of steroid-related ONFH [10, 29].
Studies have shown that quantitative analysis of cytosine methylation can identify molecular markers in diseases [30]. Specific hypermethylated CpG sites can be used to diagnose diseases [31, 32]. To further study the applicability of OPG, RANK, and RANKL gene methylation levels as potential diagnostic biomarkers for steroid-related ONFH, ROC analysis was performed. The results showed that the AUC values of 3 CpG units in the OPG gene, 15 CpG units in the RANK gene, and 10 CpG units in the RANKL gene were greater than 0.5. Among them, RANK2_CpG_10.11, RANK2_CpG_16, RANK2_CpG_23.24.25.26.27, RANK3_CpG_8, RANKL1_CpG_1, RANKL1_CpG_25.26.27, RANKL1_CpG_44.45, and RANKL2_CpG_9.10.11.12 are the CpG units with the most diagnostic value, which may have a certain significance for the early prediction of steroid-related ONFH.
In this experiment, we selected the patient’s blood for testing, not bone tissue. Is there a difference in methylation between the two? Ebrahimi P et al. [33] believed that peripheral blood is a viable substitute for bone tissue in DNA methylation studies. However, Walton E et al. [34] suggested that there was a lack of correspondence between DNA methylation in blood and tissues. Most DNA methylation markers in peripheral blood cannot reliably predict DNA methylation status. In this experiment, the sample we selected was blood, which may not be as sensitive as femoral head tissue in investigating the cause of steroid-related ONFH.
Methylation itself is a reversible chemical modification of DNA. Methylated cytosine can be converted back into cytosine through active or passive demethylation. Given this characteristic, we not only hope to diagnose the disease by identifying DNA methylation, but also wish to treat the disease by changing the DNA methylation. Therefore, can diseases be treated by changing the status of DNA methylation? Some studies have shown that regulating the degree of methylation of the target gene, has a positive therapeutic effect on diseases. For example, DNA methylation inhibitors have become the principal means to treat certain haematological malignancies (such as myelodysplastic syndrome, chronic myelomonocytic leukaemia, and acute myeloid leukemia) [35, 36]. Some drugs can treat kidney disease by inhibiting DNA methylation (such as 5-azacytidine and decitabine) or activating DNA demethylation (such as hydralazine) [37]. In orthopaedic diseases, DNA methylation is also expected to become a future treatment and diagnosis target for RA [38]. The author believes that the treatment of disease by changing the DNA methylation status is very promising. However, how DNA methylation acts on related proteins in steroid-related ONFH and treats the disease still requires in-depth exploration and further research.
It must be pointed out that our research has certain limitations. First, in this study, only an association between DNA methylation and steroid-related ONFH was studied, not causality. In particular, we did not detect the expression levels of the three genes. The results of DNA methylation analysis by MassARRAY require further validation. Second, abnormal DNA methylation is not confined to CpG islands. Methylation changes in other sites (such as the CpG shore and CpG shelve) should not be ignored. Third, the small sample size of our research limits the generalization of the results. In subsequent studies, we will use more reasonable test methods on larger samples to verify the above results.
In summary, our results show that the methylation status of the OPG, RANK, and RANKL genes in steroid-related ONFH patients is different from that of normal controls. The hypermethylation of some CpG units increases the risk of steroid-related ONFH. Detecting the methylation of the CpG sites of the above genes, it has certain significance for the early diagnosis of steroid-related ONFH.