MiR-125b Regulates Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells By Targeting Smad4

Objective: To investigate whether miR-125b regulates bone marrow mesenchymalstem cells (MSCs) osteogenic differentiation by targeting Smad4.Methods: The density gradient centrifugation method was used to isolate and culturethe bone marrow MSCs. The cell surface markers of MSCs were analyzed using flowcytometry. Moreover, MSCs osteogenic differentiation was determined by ALPactivity and the expression levels of osteogenic specific genes such as RUNX2andOsterix. MSCs were transfected with miR-125b mimics or inhibitors, followed byinduction of osteogenic differentiation, then the effects of miR-125b on MSCsosteogenic differentiation was determined. Smad43’UTR-luciferase vector wasconstructed and contransfected with miR-125b into COS7cells. After48h,dual-luciferase reporter gene assay was employed to examine the effect of miR-125bon luciferase activity. MSCs were transfected with miR-125b mimics and induced intoosteogenic differentiation. Then, qRT-PCR and Western blotting assays were used todetect the expressions of Smad4mRNA and protein. MSCs were inducted intoosteoblasts after transfecting with Smad4siRNA, and the effect of Smad4knockdownon osteogenic differentiation was observed.Results: The cell surface markers CD34and CD45were negative but CD29andCD44positive in MSCs cells, which is consistent with bone marrow mesenchymalstem cells in basic features. The ALP activity and the expression of osteogenicspecific genes such as RUNX2and Osterix were markly upregulated in MSCscultured in osteogenic differentiation medium, suggesting MSCs could differentiateinto osteoblasts. Overexpression of miR-125b inhibits MSCs osteogenicdifferentiation, however miR-125b knockdown promoted the process. Wedemonstrated miR-125b could bind to the Smad43’UTR and inhibited the luciferaseactivity. Smad4mRNA and protein expressions were significantly down-regulated inMSCs inducted into osteogenic differentiation when miR-125b overexpressed. The knocknown of Smad4could suppress ALP activity and the expressions of RUNX2and Osterix, indicating that Smad4siRNA simulates at least in part the function ofmiR-125b as regulator of MSCs osteogenic differentiation.Conclusion:(1) Human MSCs can be isolated from human bone marrow withdensity gradient centrifugation.human and be induced into osteoblasts in vitro.(2) miR-125b can suppresses MSCs osteogenic differentiation by directly targetingSmad4.