MiR-26b-5p Promotes Osteogenesis of Bone Mesenchymal Stem Cells via Suppressing FGF21
Abstract Introduction: It has been established that miR-26b-5p actively participate in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs), which is of great value in osteoporosis treatment. Database showed that Fibroblast growth factor(FGF)21 is a potential binding site of miR-26b-5p. This study aimed to investigate the molecular osteogenic mechanisms of miR-26b-5p targeting FGF21 in postmenopausal osteoporosis (PMOP). Methods: 5ml of bone marrow was aspirated from the anterior superior iliac spine in 10 PMOP women during bone marrow puncture. BMSCs were used to establish an in vitro cell model, and BMSCs markers were analyzed by flow cytometry. miR-26b-5p and FGF21 were overexpressed for 48h, and then placed in an osteogenic induction medium for osteogenic induction culture, the expression of RNA was detect using RT-qPCR. Cells from miR-26b-5p group were collected on days 7, 14 and 21 of induction for ALP and alizarin red S staining. On day 7 of induction, RT-qPCR was used to measure Runx2, Osterix (Osx), and target gene FGF21 expression levels in each group. The dual-luciferase reporter gene system was used to verify that FGF21 was a direct target of miR-26b-5p. FGF21 was measured by western blotting in the miR-26b-5p overexpression group and in the miR-26b-5p inhibition group. Results: BMSCs were identified according with the antigenic characteristics. miR-26b-5p expression was significantly upregulated after the expression of miR-26b-5p mimics; however, FGF21 expression was downregulated after FGF21 mimics. After overexpression of miR-26b-5p, the alkaline phosphatase activity and nodules of alizarin red S in the culture medium gradually increased as the induction time increased. RT-qPCR showed that the expressions of master osteogenic factors Runx2 and Osx in the BMSC+ osteogenic differentiation medium group was significantly higher than in the BMSC group, the expressions of the factors in the BMSC+ miR-26b-5p overexpression group was significantly higher than in the control group. Target gene FGF21 expression was significantly lower in the BMSC+ osteogenic differentiation medium group than in the BMSC group, and was significantly lower in the BMSC+ miR-26b-5p overexpression group than in the control group. Luciferase reporter assays demonstrated that FGF21 was a direct target of miR-26b-5p. Finally, western blotting analysis showed that FGF21 expression was significantly downregulated in the miR-26b-5p overexpressed group and upregulated in the miR-26b-5p inhibition group. Conclusion: miR-26b-5p can regulate the osteogenic differentiation of BMSCs and participate in PMOP pathogenesis via suppressing FGF21. The present study provides the basis for further studies on PMOP.