Department of Hematology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Immunology, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
J Cell Physiol. 2019 May;234(5):6230-6243. doi: 10.1002/jcp.27352. Epub 2018 Sep 24.
Osteoblastic differentiation of mesenchymal stem cells (MSCs) is the principal stage during the restoration and regeneration of bone tissue. Epigenetic modifications such as DNA methylation play a key role in the differentiation process of stem cells. In this study, the methylation status of the promoter region of ZBTB16 and Twist1 genes and their role in controlling osteoblastic differentiation in MSCs was investigated during the osteoblastic differentiation of MSCs.
The MSCs were cultured under standard conditions and differentiated into the osteoblasts. We had three treatment groups including 5-azacytidine (methylation inhibitor), metformin (Twist-inhibitor), and procaine (Wnt/β-catenin inhibitor) and a non-treated group (control). Methylation level of DNA in the promoter regions was monitored by methylation specific-quantitative polymerase chain reaction (PCR). Also, the mRNA levels of key genes in osteoblastic differentiation were measured using real-time PCR.
ZBTB16 gene expression was upregulated, and promoter methylation was decreased. For Twist1 messenger RNA (mRNA) level decreased and promoter methylation increased during osteoblastic differentiation of MSCs. 5-Azacytidine caused a significant reduction in methylation and increased the mRNA expression of ZBTB16 and Twist1. Metformin repressed the Twist1 expression, and therefore osteoblastic differentiation was increased. On the opposite side, procaine could block the WNT/β-catenin signaling pathway, as a consequence the gene expression of key genes involved in osteoblastic differentiation was declined.
We found that methylation of DNA in the promoter region of ZBTB16 and Twist1 genes might be one of the main mechanisms that controlling the gene expression during osteoblastic differentiation of MSCs. Also, we could find an association between regulation of Twist1 and ZBTB16 genes and osteoblastic differentiation in MSCs by showing the relation between their expression and some key genes involved in osteoblastic differentiation. In addition, we found a connection between the Twist1 expression level and osteoblastic differentiation by using a Twist-inhibitor (metformin).
间充质干细胞（MSCs）的成骨细胞分化是骨组织修复和再生的主要阶段。表观遗传修饰，如 DNA 甲基化，在干细胞的分化过程中起着关键作用。在这项研究中，我们研究了 MSCs 成骨细胞分化过程中 ZBTB16 和 Twist1 基因启动子区域的甲基化状态及其对 MSC 成骨细胞分化的控制作用。
在标准条件下培养 MSCs 并诱导其向成骨细胞分化。我们有三个处理组，包括 5-氮杂胞苷（甲基化抑制剂）、二甲双胍（Twist 抑制剂）和普鲁卡因（Wnt/β-连环蛋白抑制剂）以及未经处理的组（对照组）。通过甲基化特异性定量聚合酶链反应（PCR）监测 DNA 启动子区域的甲基化水平。此外，还使用实时 PCR 测量成骨分化过程中关键基因的 mRNA 水平。
ZBTB16 基因表达上调，启动子甲基化减少。在 MSCs 成骨分化过程中，Twist1 信使 RNA（mRNA）水平降低，启动子甲基化增加。5-氮杂胞苷导致甲基化显著减少，ZBTB16 和 Twist1 的 mRNA 表达增加。二甲双胍抑制 Twist1 表达，从而增加成骨分化。相反，普鲁卡因可以阻断 WNT/β-连环蛋白信号通路，导致参与成骨分化的关键基因的表达下降。
我们发现，ZBTB16 和 Twist1 基因启动子区域的 DNA 甲基化可能是控制 MSCs 成骨分化过程中基因表达的主要机制之一。此外，我们通过显示 Twist1 和 ZBTB16 基因表达与成骨分化相关的一些关键基因之间的关系，发现了它们在 MSCs 成骨分化中的调控之间的关联。此外，我们通过使用 Twist 抑制剂（二甲双胍）发现了 Twist1 表达水平与成骨分化之间的联系。
