Abstract:

Objective To explore the expression and biological effect of miR-424 during osteogenic differentiation of human adipose derived mesenchymal stem cells (hASC). Methods The expressions of miR-424 at different stages of osteogenic differentiation of hASC were detected with real-time PCR. miR-424 was over-expressed or miR-424 expression was inhibited via infection of hASCs with lentivirus particles in vitro. Western blotting, immunocytochemistry, and alkaline phosphatase (ALP) staining and alizarin red (ARS) staining were performed. The effects of miR-424 on osteogenic differentiation of hASCs induced with BMP-2 and osteogenic differentiation medium were investigated. The target gene of miR-424 was predicted and was verified with qPCR and Western blotting. Results The results of qPCR revealed that the expression of miR-424 declined with the time of osteogenic differentiation (P<0.05). The protein levels of ALP and osteopontin (OPN) decreased in the over-expression group and increased in the inhibition group with the induction of bone morphogenetic protein 2(BMP-2) (P<0.05). The results of immunocytochemistry revealed that over-expression of miR-424 significantly reduced the cell positive rate of bone sialoprotein (BSP)(P<0.05). ALP and ARS staining showed that the activity of ALP and capacity of mineralization in the over-expression group decreased, while the activity of ALP and capacity of mineralization increased in the inhibition group. Over-expression of miR-424 could decrease the mRNA and protein expression levels of Smad5 in hASCs and inhibition of miR-424 could increase the mRNA and protein expression levels of Smad5 (P<0.05). Conclusion The expression of miR-424 declines during the osteogenic differentiation of hASCs and miR-424 inhibits the osteogenic differentiation of hASCs.

Key words: human adipose derived mesenchymal stem cells, miR-424, Smad5, osteogenic differentiation