Objectives: Elucidate the role of miRNA-218 on RUNX2 in DSC under osteoinductive conditions.
Methods: Tissue samples of periodontal ligament, dental pulp, and attached gingiva were harvested from healthy discarded 3rd molars. Cells were FACS sorted to obtain a STRO-1+ enriched cell population. Total RNA was extracted at day 0 (D0) to profile miRNA expression and qRT-PCR was performed to demonstrate OCT4A/NANOG activity. DSC were cultured for 0, 7, 14, 21 and 28 days under osteogenic conditions or control. qRT-PCR was performed at each time-point to evaluate RUNX2 activity. When RUNX2 reached the highest expression, total RNA was collected for miRNA analysis. Alizarin red staining was performed at D28 to confirm mineralization. qRT-PCR and Western Blot tests were used to confirm the correlation between hsa-mir-218 and RUNX2. Human bone marrow stem cells were used as positive control for all the experiments.
Results: qRT-PCR results from OCT4A/NANOG confirmed undifferentiated status of DSC at baseline. qRT-PCR results for RUNX2 demonstrated osteogenic induction, which was confirmed using alizarin red. Microarray of 765 miRNA indicated a 3-6 decrease in hsa-mir-218 in all DSC upon differentiation. qRT-PCR and Western Blot demonstrated an increase in RUNX2 associated to hsa-mir-218 downregulation.
Conclusion: In the process of DSC differentiation, MiRNA-218 upregulates RUNX2 by targeting the 3′-untranslated region of the mRNA.
Keywords: Cell biology, Gene expression, MicroRNA, Mineralization and Regeneration