International endodontic journal · 2018
Inflammation-induced overexpression of microRNA-223-3p regulates odontoblastic differentiation of human dental pulp stem cells by targeting SMAD3.
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Huang X, Liu F, Hou J, Chen K
Аннотация
AIM: 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.
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 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.
CONCLUSIONS: miR-223-3p is implicated in the regulation of odontoblast differentiation, which may be involved in the process of pulpitis repair.
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 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.
CONCLUSIONS: miR-223-3p is implicated in the regulation of odontoblast differentiation, which may be involved in the process of pulpitis repair.