MiR-21 Synergizes With BMP9 In Osteogenic Differentiation By Activating BMP9/smad Signaling Pathway In Mesenchymal Stem Cells

Objective: to investigate if there is a crosstalk between BMP9 and miR-21,and if miR-21 is a downsteam target of BMP9; to investigate the effect of miR-21 and antimiR-21 on BMP9-induced osteogenic differentiation of mesenchymal stem cells.to illuminate the target gene of miR-21 in BMP9-induced osteogenic differentiation and detect the effect of target gene in BMP9-induced osteogenic differentiation.Methods: using Ad-BMP9 infecting MEFs,C2C12 cells,C3H10 T1/2 cells,we detect the expression of miR-21 at 0,1,3,5,7,9 days by q-PCR.After transferring miR-21 mimic 24 hours,the expression of BMP were testing by q-PCR.At different passage of HMSC,the expression of miR-21 was detected by Q-PCR.In vitro,we test if miR-21 and antimiR-21 can affect BMP9-induced ALP activity and the calcium deposition.What’more,we monitor the expression of BMP9 downstream factors p-Smad1/5,Runx2 by western blot. To investigate the effect of miR-21 and antimiR-21 on BMP9-induced osteogenic differentiation of mesenchymal stem cells. we detected the level of p-Smad1/5 and Runx2 by Western blot at the same time.Target prediction tools were used to search possible target genes for miR-21 taking in BMP9 induced ostegenic differentiation. By dual luciferase assay,we clear and definite the drect target effect of possible target gene and miR-21.Lastly,we explicit the influence of downregulation target gene in BMP9-induced osteogenic differentiation by si RNA of target gene.Results: The level of miR-21 expression was about 3-fold higher than that in control groups of C2C12 cells and 5-fold in i MEF cells and 2-fold in C3H10 T1/2 cells.At the 2,3,3passages of HMSC,the expression of miR-21 were up-regulation.MiR-21 can potentiate BMP9-induced ALP activity and the calcium deposition. The levels of p-Smad1/5 and Runx2 were upregulated at the same conditions.MiR-21 can regulation the m RNA and protein expression of Smad7.MiR-21 can decrease the luciferase activity of Smad7.Conclusions: MiR-21 was significantly upregulated by BMP9 during the osteogenesis of mesenchymal stem cells. MiR-21 can potentiate BMP9-induced osteoblast lineage commitment.The high level of miR-21 was significant for sustaining BMP9-Smad Signaling activity during osteogenesis. MiR-21 can target Smad7 derectly.