Department of Biology, Faculty of Sciences, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran.
Cancer Gene Therapy Research Center, Zanjan University of Medical Science, Zanjan, Iran.
J Oral Biosci. 2020 Dec;62(4):349-356. doi: 10.1016/j.job.2020.08.001. Epub 2020 Aug 22.
Bone marrow derived mesenchymal stem cells (BMSCs) are an irresistible choice for use in stem cell therapy and regenerative medicine. BMSCs osteoblastic differentiation is also important in bone development, diseases, malignancies, and cancers studies. Wnt signaling pathway antagonists, Dickkopf-1 (Dkk 1), Secreted Frizzled-Related Proteins (sFRPs), and Wnt Inhibitory Factor 1 (Wif1) play important roles in inducing osteoblastic differentiation. This study is the first to investigate the association between DNA methylation and gene expression of Dkk1, sFRP2, sFRP4, and Wif1 during BMSCs osteoblastic differentiation.
Human BMSCs were isolated and characterized using flow cytometry. Then, cells were treated with osteo-differentiation medium for three weeks. Alizarin red S staining and polymerase chain reaction (PCR) (alkaline phosphatase/osteocalcin) were performed for confirmation. The expression of Dkk 1, sFRP2, sFRP4, and Wif1 genes was evaluated at days 7, 14, and 21 using real-time PCR. Methylation-specific PCR (MSP) was performed to detect the methylation status of the promoters of the genes.
Data showed significant decreases (P < 0.05) during various days of BMSCs differentiation, while the promoters of the genes remained mostly un-methylated.
The down-regulation of Dkk 1, sFRP2, sFRP4, and Wif1 regulates various stages of human BMSCs during osteoblastic differentiation. DNA methylation does not interfere in the down-regulation of these genes, except for Wif1. We propose that the Wnt antagonist gene promoters should remain un-methylated during osteoblastic differentiation of BMSCs and that the down-regulation of these genes may contribute to other epigenetic mechanisms, other than DNA methylation, which implicitly indicates the role of DNA methylation in osteogenic cancers.
骨髓间充质干细胞（BMSCs）是干细胞治疗和再生医学中不可抗拒的选择。BMSCs 的成骨分化在骨骼发育、疾病、恶性肿瘤和癌症研究中也很重要。Wnt 信号通路拮抗剂 Dickkopf-1（Dkk1）、分泌型卷曲相关蛋白（sFRPs）和 Wnt 抑制因子 1（Wif1）在诱导成骨分化中发挥重要作用。本研究首次探讨了在 BMSCs 成骨分化过程中 Dkk1、sFRP2、sFRP4 和 Wif1 的 DNA 甲基化与基因表达之间的关系。
使用流式细胞术分离和鉴定人 BMSCs。然后，用成骨分化培养基处理细胞 3 周。用茜素红 S 染色和聚合酶链反应（PCR）（碱性磷酸酶/骨钙素）进行确认。在第 7、14 和 21 天用实时 PCR 评估 Dkk1、sFRP2、sFRP4 和 Wif1 基因的表达。用甲基化特异性 PCR（MSP）检测基因启动子的甲基化状态。
数据显示，在 BMSCs 分化的不同天数，Dkk1、sFRP2、sFRP4 和 Wif1 的表达显著降低（P<0.05），而基因启动子大多保持非甲基化状态。
Dkk1、sFRP2、sFRP4 和 Wif1 的下调调节了人 BMSCs 成骨分化的各个阶段。DNA 甲基化不干扰这些基因的下调，除了 Wif1。我们提出，在 BMSCs 成骨分化过程中，Wnt 拮抗剂基因启动子应保持非甲基化状态，这些基因的下调可能与 DNA 甲基化以外的其他表观遗传机制有关，这暗示了 DNA 甲基化在成骨性癌症中的作用。
