Expression of Sclerostin in Osteoporotic Fracture Patients Is Associated with DNA Methylation in the CpG Island of the SOST Gene

Abstract

Purpose: SOST gene is one of the key factors in regulating bone absorption. Although there are reports showing diverse transcription factors, epigenetic modification could be responsible for regulating SOST gene expression. There is still little exploration on promoter methylation status of SOST gene in osteoporotic bone tissues. The aim of this study is to investigate the involvement of CpG methylation in regulation of SOST expression in patients with primary osteoporosis.

Methods: The diagnosis of osteoporosis was established on the basis of dual energy X-ray absorptiometry to measure BMD. All femoral bone tissues were separated in surgeries. After extracting total RNA and protein, we checked the relative expression levels of SOST by quantitative real-time PCR and western blot. Also, immunohistochemical staining was performed to observe the expression of SOST protein in the bone samples. The genomic DNA of non-OPF (non-osteoporotic fracture bone tissues) and OPF (osteoporotic fracture bone tissues) were treated by bisulfite modification, and methylation status of CpG sites in the CpG island of SOST gene promoter was determined by DNA sequencing.

Results: SOST gene expression in the non-OPF group was lower than that in OPF group. Bisulfite sequencing result showed that SOST gene promoter was slightly demethylated in the OPF group, as compared with non-OPF group.

Conclusion: Our study demonstrated that DNA methylation influenced the transcriptional expression of SOST gene, which probably may play an important role in the pathogenesis of primary osteoporosis.

Figure 1

Expression level of SOST in bone tissue samples. (a) Total RNA was extracted from bone tissues of patients with OPF or non-OPF. GAPDH was used as an internal control. The data are expressed as mean ± SD (n = 16). ^∗ p < 0.05. (b) Total proteins extracted from bone tissues of patients with OPF or non-OPF were analyzed by western blot using anti-SOST antibody. β-Actin was used as loading control (n = 3). (c) The protein levels of SOST in control and OPF groups were quantified using ImageJ software. Data is presented as mean ± SD (n = 3, p < 0.05).

Figure 2

Detection of SOST in bone samples by immunohistochemical staining. Bone samples of OPF and non-OPF were decalcified and sectioned. Antisclerostin antibody was used for immunohistochemical staining. SOST was specifically expressed in osteocytes. (a) The number of SOST-positive osteocytes was counted. Data is presented as mean ± SD (n = 3, p < 0.05). (b) Typical images of immunohistochemical staining of SOST in control and OPF groups.

Figure 3

Schematic figure indicates 16 CpG sites in CpG island of the SOST gene promoter. Exons in upper case, everything else in lower case. The CpG sites were shown in green.

Figure 4

Epigenetic regulation of SOST in bone tissues. DNA methylation status of SOST promoter in three non-OPF and three OPF samples using sodium bisulfite sequencing. Each PCR product was subcloned and subjected to nucleotide sequencing analysis. (a) The percentage of methylated CpG sites in SOST promoter was calculated based on the BSP sequencing result. (b) BSP sequencing result of methylated CpG sites in each samples. Sequenced clones were depicted by filled (methylated) and open (unmethylated) circles for each CpG site.