Backgrounds: MicroRNAs play diverse roles in a range of biological processes, including osteoblastic differentiation. The tumor suppressor miR-219a-5p has been found to be functionally important in several tumor types, yet its expression and mechanistic relevance in the context of bone marrow stem cell (BMSC) osteogenic differentiation remain to be defined.
Methods: Osteogenic induction medium was used to induce osteogenic differentiation in human BMSCs (hBMSCs), after which alkaline phosphatase (ALP) activity was measured and Alizarin red S staining was performed to gauge the efficiency of osteogenesis. Additionally, qPCR was used to assess miR-219a-5p, GSK-3β, and osteogenic gene expression, while levels of proteins associated with osteoblastic differentiation and the Wnt/β-catenin signaling pathway were detected via Western blotting in order to assess how miR-219a-5p inhibitors and mimics influenced the processes of osteogenic differentiation. Mir-219a-5p and GSK-3β binding sites were predicted via a bioinformatics approach, with dual-luciferase reporter assays being performed to validate such binding.
Results: A time-dependent increase in miR-219a-5p expression was observed over the course of hBMSC osteogenic differentiation. Transfection with miR-219a-5p mimics enhanced the expression of markers of osteoblast cells (RUNX2, ALP), whereas miR-219a-5p inhibitors yielded the opposite effect. The ability of miR-219a-5p to directly bind the 3’-UTR of GSK-3β was confirmed via luciferase reporter assay, highlighting GSK-3β as a target of this miRNA involved in promoting osteogenesis such that miR-219a-5p is able to regulate osteogenic differentiation in BMSCs via the GSK-3β/β-catenin pathway. Moreover, we found that overexpressing GSK-3β was sufficient to reverse the impact of miR-219a-5p on hBMSC osteogenesis.
Conclusion: These data indicate that miR-219a-5p is capable of directly targeting the Wnt/β-catenin pathway gene GSK-3β, thereby regulating BMSC osteogenesis. As such, targeting this miRNA may represent a new approach to enhancing hBMSC osteogenesis.