Purpose: To explore the effect of enamel matrix proteins(EMPs) on osteogenesis and adipogenesis of stem cells from human exfoliated deciduous teeth SHED), and explore its molecular mechanism.
Methods: SHEDs were used to detect the expression of its surface antigens CD73, CD146, CD34 and CD45 by flow cytometry. SHED was induced by OB osteogenic induction liquid, and then the osteogenic differentiation ability was measured by alizarin red staining. SHEDs were divided into 4 groups, NC group had invalid sequence shRNA interfered with SHED, EMPs group had invalid sequence shRNA interfered with SHED. Then 100 μg/L EMPs was used to interfere with SHED. In miR-32 inhibitor group, miR-32 shRNA plasmid was used to interfere with SHED; while in EMPs+miR-32 inhibitor group, 100 μg/L EMP was used to intervene SHED after silencing miR-32. QPCR was used to detect the expression of miR-32, dentin sialophosphoprotein (DSPP), dentin matrix protein 1, DMP-1, peroxisome proliferators-activated receptor γ (PPARγ) and CCAAT enhancer binding protein α (C/EBPα) gene expression; Western blot was used to detect the expression of DSPP, DMP-1, PPARγ and C/EBPα protein expression; Alizarin red staining was used to detect SHED osteogenic capacity; Oil red O staining was used to detect adipogenetic capacity of SHED.
Results: The results of flow cytometry showed that SHED had positive expression of CD146 and CD73, and negative expression of CD34 and CD45, which was consistent with the characteristics of stem cell surface markers. Alizarin red staining and oil red O staining showed mineralized nodules and oil droplets increased significantly, consistent with the multi-directional differentiation characteristics of stem cells. Compared with NC group, the expression of miR-32 gene in EMPs group was significantly increased(P<0.05), and the expression of miR-32 in miR-32 inhibitor group and EMPs+miR-32 inhibitor group was significantly decreased(P<0.05). Compared with NC group, the expression of DSPP and DMP-1, the number of mineralized nodules in EMPs group were significantly increased(P<0.05), the expression of PPARγ and C/EBPa and the number of lipid droplets were significantly decreased (P<0.05), while the result of miR-32 inhibitor group was the opposite (P<0.05). Compared with miR-32 inhibitor group, there was no significant difference in the expression of DSPP, DMP-1, PPARγ and C/EBPα, number of mineralized nodules and oil droplets in EMPs+miR-32 inhibitor group(P>0.05). Compared with EMPs group, the expression of DSPP and DMP-1 and the number of mineralized nodules in EMPs+miR-32 inhibitor group were significantly reduced(P<0.05), while the expression of PPARγ and C/EBPα and the number of lipid droplets were significantly increased(P<0.05).
Conclusions: EMPs can regulate osteogenic and adipogenic differentiation of SHED by promoting the expression of miR-32.