Inflammation-induced overexpression of microRNA-223-3p regulates odontoblastic differentiation of human dental pulp stem cells by targeting SMAD3.

AIM: To profile miRNA expression between inflamed and healthy human dental pulp tissues and to investigate how the up-regulation of miR-223-3p in the inflamed pulp tissue regulates odontoblast differentiation and regeneration.

METHODOLOGY: Microarray analysis was used to identify differences in miRNA expression patterns between healthy and inflamed pulp tissue. The results were validated using quantitative real-time PCR. To determine the effect of miR-223-3p on odontoblast differentiation, miR-223-3p was overexpressed in human dental pulp stem cells (DPSCs), which were cultured in mineralizing induction medium (to induce odontoblast differentiation). To identify the target genes of miR-223-3p, an SABiosciences Human Osteogenesis PCR Array, combined with bioinformatics, was used. Furthermore, a dual-luciferase reporter assay and a small interfering RNA (siRNA) experiment were used to confirm the relationship between miR-223-3p and its target gene. Statistical analysis was performed using the Student's t test or one-way analysis of variance (ANOVA); P<0.05 was considered statistically significant.

RESULTS: Seventy-nine miRNAs were significantly differentially expressed (fold change >2.0; P<0.05) between the two tissues. In particular, miR-223-3p was markedly up-regulated in inflamed dental pulp. Overexpression of miR-223-3p in DPSCs significantly increased the protein levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP-1) (P<0.05). However, the SMAD family member 3 (SMAD3) protein level was significantly lower than in control DPSCs (P<0.05). Bioinformatics and the dual-luciferase assay reporter assay indicated that Smad3 was a potential target of miR-223-3p. Knockdown of Smad3 in DPSCs subjected to mineralization induction resulted in detection of DSPP and DMP-1 earlier than in control DPSCs, and it increased the protein level of alkaline phosphatase (ALP), thereby promoting odontoblast differentiation.

CONCLUSIONS: miR-223-3p is implicated in the regulation of odontoblast differentiation, which may be involved in the process of pulpitis repair. This article is protected by copyright. All rights reserved.