Sevda Rahimzadeh
1,2 
, Reza Rahbarghazi
1, Somayeh Aslani
2, Hadi Rajabi
2, Zeinab Latifi
2, Majid Farshdousti Hagh
3, Alireza Nourazarian
2, Hojjatollah Nozad Charoudeh
4, Mohammad Nouri
5* , Alireza Abhari
1,2* 1 Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
3 Department of Hematology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
4 Anatomical Sciences Department, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
5 Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Introduction: Nowadays, mesenchymal stem cells are touted as suitable cell supply for the restoration of injured bone tissue. The existence of osteogenic differentiation makes these cells capable of replenishing damaged cells in the least possible time. It has been shown that epigenetic modifications, especially DNA methylation, contribute to the regulation of various transcription factors during phenotype acquisition. Hence, we concentrated on the correlation between the promoter methylation and the expression of genes DLX3, ATF4, and FRA1 during osteoblastic differentiation of adipose-derived mesenchymal stem cells in vitro after 21 days.
Methods: Adipose-derived mesenchymal stem cells were cultured in osteogenesis differentiation medium supplemented with 0.1 µM dexamethasone, 10 mM β-glycerol phosphate, and 50 µM ascorbate-2-phosphate for 21 days. RNA and DNA extraction was done on days 0, 7, 14, and 21. Promoter methylation and expression levels of genes DLX3, ATF4, and FRA1 were analyzed by methylation-specific quantitative PCR and real-time PCR assays, respectively.
Results: We found an upward expression trend with the increasing time for genes DLX3, ATF4, and FRA1 in stem cells committed to osteoblast-like lineage compared to the control group (P<0.05). On the contrary, methylation-specific quantitative PCR displayed decreased methylation rates of DLX3 and ATF4 genes, but not FRA1, over time compared to the non-treated control cells (P<0.05). Bright-field images exhibited red-colored calcified deposits around Alizarin Red S-stained cells after 21 days compared to the control group. Statistical analysis showed a strong correlation between the transcription of genes DLX3 and ATF4 and methylation rate (P<0.05).
Conclusion: In particular, osteoblastic differentiation of adipose-derived mesenchymal stem cells enhances DLX3 and ATF4 transcriptions by reducing methylation rate for 21 days.