| Abstract
Mesenchymal stem cells (MSCs) are multipotent stem cells and show distinct features
such as capability for self‐renewal and differentiation into several lineages of cells
including osteoblasts, chondrocytes, and adipocytes. In this study, the methylation
status of the promoter region of zinc finger and BTB domain containing 16 (ZBTB16),
twist‐related protein 1(Twist1), de novo DNA methyltransferases 3A (DNMT3A),
SRY‐box 9 (Sox9), osteocalcin (OCN), and peroxisome proliferator‐activated receptor
γ2 (PPARγ2) genes and their messenger RNA (mRNA) expression levels were
evaluated during the osteoblastic differentiation of MSCs (ODMSCs). We planned
two experimental groups including zoledronic acid (ZA)‐treated and nontreated cells
(negative control) which both were differentiated into the osteoblasts. Methylation
level of DNA in the promoter regions was assayed by methylation‐specificquantitative
polymerase chain reaction (MS‐qPCR), and mRNA levels of the target
inhibitory/stimulatory genes during osteoblastic differentiation of MSCs were
measured using real‐time PCR. During the experimental induction of ODMSCs, the
mRNA expression of the OCN gene was upregulated and methylation level of its
promoter region was decreased. Moreover, Sox9 and PPARγ2 mRNA levels were
attenuated and their promoter regions methylation levels were significantly
augmented. However, the mRNA expression of the DNMT3A was not affected
during the ODMSCs though its methylation rate was increased. In addition, ZA could
enhance the expression of the ZBTB16 and decrease its promoter regions
methylation and on the opposite side, it diminished mRNA expression of Sox9,
Twist1, and PPARγ2 genes and increased their methylation rates. Intriguingly, ZA did
not show a significant impact on gene expression and methylation levels the OCN and
DNMT3A. We found that methylation of the promoter regions of Sox9, OCN, and
PPARγ2 genes might be one of the main mechanisms adjusting the genes expression
during the ODMSCs. Furthermore, we noticed that ZA can accelerate the MSCs
differentiation to the osteoblast cells via two regulatory processes; suppression of osteoblastic differentiation inhibitor genes including Sox9, Twist1, and PPARγ2, and
through promotion of the ZBTB16 expression. |