IADR Abstract Archives
Dose-dependent pharmacological inhibition of miR-205 in human dental pulp stem cells promotes the expression of mineralisation-associated markers
Objectives: Our previous RNAseq analysis of epigenetically-driven dental pulp cell (DPC) mineralisation identified several significantly altered miRNA in rat and human DPC populations, in particular miR-205. The objectives of the current study are to optimise the concentration for pharmacological gymnotic inhibition of miR-205 in human dental pulp stem cells (hDPSCs) in vitro, and to investigate the effects of varying concentrations on expression of validated mineralisation-associated miR-205 target genes and on hDPSC-viability.
Methods: hDPSCs were continuously cultured with varying concentrations (0.1µM, 0.2µM and 1µM) of a pharmacological miR-205 inhibitor for 14 days. A negative control bearing no resemblance to any known human miRNA was used for comparison. Effective inhibition of miR-205 was confirmed by qRT-PCR quantification of previously validated target genes of miR-205, including the mineralisation-associated genes RUNX2, SMAD1 and SMAD4 at days 4 and 14. The effect of the inhibitor on cell viability was investigated using an MTT assay at 48 h.
Results: Inhibitor treatment did not affect DPC viability at all tested concentrations (0.1µM, 0.2µM and 1µM). On day 4, expression of RUNX2 was increased in DPCs treated with 0.1µM (2.1-fold). At the same timepoint, there was no change in expression of RUNX2 in DPCs treated with 0.2µM or 1µM inhibitor. The expression of SMAD1 was inversely proportional to the inhibitor concentration, demonstrating a 1.7-fold increase in the 0.1µM group, a 1.6-fold increase in the 0.2µM group, and a 1.4-fold increase in the 1µM group. The expression of SMAD4 demonstrated a similar peak in the 0.1µM group (2-fold), with lower expression observed in the 0.2µM and 1µM groups (1.1- and 1.2-fold, respectively).
Conclusions: The optimal inhibitor concentration for miR-205 in hDPSCs was demonstrated by the increase in expression of three target genes with no adverse effect on DPSC viability. MiR-205 inhibition stimulates the expression of mineralisation-associated genes and presents opportunities for therapeutic application in vital pulp treatment.
Methods: hDPSCs were continuously cultured with varying concentrations (0.1µM, 0.2µM and 1µM) of a pharmacological miR-205 inhibitor for 14 days. A negative control bearing no resemblance to any known human miRNA was used for comparison. Effective inhibition of miR-205 was confirmed by qRT-PCR quantification of previously validated target genes of miR-205, including the mineralisation-associated genes RUNX2, SMAD1 and SMAD4 at days 4 and 14. The effect of the inhibitor on cell viability was investigated using an MTT assay at 48 h.
Results: Inhibitor treatment did not affect DPC viability at all tested concentrations (0.1µM, 0.2µM and 1µM). On day 4, expression of RUNX2 was increased in DPCs treated with 0.1µM (2.1-fold). At the same timepoint, there was no change in expression of RUNX2 in DPCs treated with 0.2µM or 1µM inhibitor. The expression of SMAD1 was inversely proportional to the inhibitor concentration, demonstrating a 1.7-fold increase in the 0.1µM group, a 1.6-fold increase in the 0.2µM group, and a 1.4-fold increase in the 1µM group. The expression of SMAD4 demonstrated a similar peak in the 0.1µM group (2-fold), with lower expression observed in the 0.2µM and 1µM groups (1.1- and 1.2-fold, respectively).
Conclusions: The optimal inhibitor concentration for miR-205 in hDPSCs was demonstrated by the increase in expression of three target genes with no adverse effect on DPSC viability. MiR-205 inhibition stimulates the expression of mineralisation-associated genes and presents opportunities for therapeutic application in vital pulp treatment.