MiR-148a-3p Regulates Stem Cell Osteogenic Differentiation and Enamel Development by Targeting Runt-Related Transcription Factor 2 and E-cadherin via the Wnt1/β-catenin Signaling Pathway

Abstract

: We aimed to evaluate the regulatory effects of miR-148a-3p on stem cell osteogenic differentiation and enamel development by targeting runt-related transcription factor 2 (RUNX2) and E-cadherin, respectively. TargetScan software was utilized to predict the binding sites between miR-148a-3p and osteogenic marker gene RUNX2 or E-cadherin. The changes in miR-148a-3p expression during osteogenic differentiation of epidermal stem cells were detected. After transfec tion with miR-148a-3p mimics or miR-148a-3p inhibitor, epidermal stem cells were induced towards osteogenic differentia tion, and the changes in RUNX2 expression were measured. The changes in miR-148a-3p expression during enamel devel opment regulated by epidermal stem cells were determined. After liposome-mediated transfection with miR-148a-3p mimics or miR-148a-3p inhibitor, epidermal stem cells were induced towards ameloblast development. Cell proliferation and apoptosis abilities were tested using methyl thiazolyl tetrazolium assay and flow cytometry, respectively. The expres sion of miR-148a-3p was detected by RT-PCR. The protein expressions of Wnt1, β-catenin, RUNX2 and E-cadherin were measured by Western blotting. Epidermal stem cells differentiated into osteoblasts through osteogenic induction culture. On 5, 12, 15 and 30 d, epidermal stem cells gradually differentiated into osteoblasts through epithelial aggregation and depres sion, mesenchymal aggregation, and dentin and enamel secretion. After transfection, compared with negative control (NC) group, the cell viability of miR-148-3p group significantly decreased (P<0.05), and the apoptosis rate increased (P<0.01). The viability of miR-148-3p inhibitor group significantly increased (P<0.05), while the apoptosis rate reduced (P<0.01). The dual-luciferase reporter assay showed that miR-148a-3p targeted Wnt1. Compared with NC group, the expression of miR-148a-3p significantly rose in miR-148a-3p group (P<0.001), and the protein expression levels of Wnt1, β-catenin, RUNX2 and E-cadherin significantly decreased. Compared with NC group, the expression of miR-148a-3p in miR-148a-3p inhibitor group significantly decreased (P<0.05), and the protein expression levels of Wnt1, β-catenin, RUNX2 and E-cad herin significantly increased. MiR-148a-3p is highly expressed in and regulates osteogenic differentiation and enamel de velopment through targeting RUNX2 and E-cadherin respectively via the Wnt1/β-catenin pathway, which plays an impor tant role in cell differentiation, stem cell proliferation and enamel development.