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

Abstract

To profile miRNA expression between inflamed and healthy human dental pulp tissues and to investigate how the upregulation of miR-223-3p in the inflamed pulp tissue regulates odontoblast differentiation and regeneration. 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. 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 upregulated 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. miR-223-3p is implicated in the regulation of odontoblast differentiation, which may be involved in the process of pulpitis repair.