Objective: We aimed to explore the role of microRNA-449b-5p in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its mechanism of action.
Materials and methods: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay was used to detect the expression levels of microRNA-449b-5p and osteogenic markers including RUNX2, OCN during BMSCs differentiation. The microRNA-449b-5p mimic and microRNA-449b-5p inhibitors were transfected into BMSCs to achieve microRNA-449b-5p overexpression and knockdown, then the expressions of osteogenic markers were detected by qRT-PCR. The ALP activity staining and the alizarin red staining were used to detect the activity of ALP and the mineralization ability of cells after overexpression and knockdown of microRNA-449b-5p. Binding sites for microRNA-449b-5p and Satb2 were predicted by TargetScan, the PicTar and microRNAanda programs, and confirmed by dual-luciferase reporter gene assay. The relationship between microRNA-449b-5p and Satb2 was analyzed by QRT-PCR and Western blot. The microRNA-449b-5p inhibitor and shSATB2 lentivirus were simultaneously transfected in BMSCs, and the expression levels of RUNX2, OCN and ALP were detected by qRT-PCR and ALP activity assays.
Results: microRNA-449b-5p expression gradually decreased during osteogenic differentiation. Overexpression of microRNA-449b-5p inhibited BMSCs differentiation by down-regulating ALP activity, RUNX2, and OCN expression, while the opposite result was observed after knockdown of microRNA-449b-5p. MicroRNA-449b-5p can bind to the 3'UTR end of Satb2, which was involved in the osteogenic differentiation of microRNA-449b-5p-regulated BMSCs, and silencing of Satb2 can abolish the positive effect of the microRNA-449b-5p inhibitor on osteoblasts differentiation.
Conclusions: microRNA-449b-5p could aggravate osteoporosis by inhibiting osteogenic differentiation of BMSCs through targeting Satb2.