miR-214 promotes periodontal ligament stem cell osteoblastic differentiation by modulating Wnt/β-catenin signaling

The canonical Wnt/beta-catenin signaling is important in the differentiation of human mesenchymal stem cells into osteoblasts. Accumulating evidence suggests that the expression of beta-catenin is, in part, regulated by specific microRNAs (miRNAs). The aim of the present study was to investigate the putative roles of miRNAs in osteoblast differentiation. Polymerase chain reaction (PCR) arrays were used to identify miRNAs that were differentially expressed between differentiated and non-differentiated periodontal ligament stem cells (PDLSCs), and reverse transcription-quantitative PCR (RT-qPCR) was used for validation. Since miR-214 was revealed to be significantly downregulated during PDLSC differentiation, its function was further investigated via silencing and overexpression. In addition, osteogenic differentiation of PDLSCs was evaluated at 10 and 21 days following induction, using Alizarin red staining and RT-qPCR analysis for mRNA expression levels of the osteogenic differentiation markers alkaline phosphatase (ALP), osteocalcin and bone sialoprotein. Furthermore, the potential target genes of miR-214 were investigated using a dual-luciferase reporter assay, RT-qPCR and western blot analysis, whereas a TOPflash/FOPflash reporter plasmid system followed by a luciferase assay was used to examine the effects of miR-214 on Wnt/beta-catenin signaling. The present results demonstrated that miR-214 was significantly downregulated during the osteoblastic differentiation of PDLSCs. Notably, its overexpression inhibited PDLSC differentiation, whereas its knockdown promoted PDLSC differentiation, as revealed by alterations in mRNA expression of osteoblast-specific genes and ALP. In addition, miR-214 was demonstrated to directly interact with the 3'-untranslated region of the beta-catenin gene CTNNB1, and suppressed Wnt/beta-catenin signaling through the inhibition of beta-catenin. The results of the present study suggested that miR-214 may participate in the regulation of the Wnt/beta-catenin signaling pathway, and may have potential as a candidate target for the development of preventive or therapeutic agents for the treatment of patients with osteogenic disorders.