Carbon Monoxide-Releasing Molecule-3 Enhances Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells via miR-195-5p/Wnt3a Pathway

These results demonstrate that CORM-3 promoted osteogenic differentiation of rat BMSCs via miR-195-5p/Wnt3a, which bodes well for the application of CORM-3 in the treatment of periodontal disease and other bone-defect diseases.

The CORM-3-stimulated osteogenic differentiation of rat BMSCs was further studied in vivo. Based on the gene sequencing experiment, the rat BMSCs were transfected with miR-195-5p mimics and inhibitor, pcDNA3.1-Wnt3a and Wnt3a siRNA. The osteogenic differentiation of rat BMSCs was measured by quantitative real-time polymerase chain reaction, Western blot and alizarin red staining. Additionally, the targeting relationship between miR-195-5p and Wnt3a was confirmed by the dual-luciferase assay.

Bone marrow-derived mesenchymal stem cells (BMSCs) are hopeful in promoting bone regeneration as their pluripotency in differentiation. Our previous study showed that carbon monoxide-releasing molecule-3 (CORM-3) increased the osteogenic differentiation of rat BMSCs in vitro. However, the mechanism remained unclear. MicroRNAs (miRNAs) play a very important role in modulating the osteogenic differentiation of BMSCs. Therefore, we researched the miRNAs involved in CORM-3-stimulated osteogenic differentiation.

MiR-195-5p was down-expressed during the CORM-3-stimulated osteogenic differentiation of rat BMSCs. CORM-3-stimulated osteogenic differentiation of rat BMSCs was inhibited with miR-195-5p overexpression, evidenced by significantly reduced mRNA and protein expressions of runt-related transcription factor 2 and osteopontin, and matrix mineralization demonstrated. On the contrary, the osteogenic differentiation was enhanced with inhibition of miR-195-5p. CORM-3-stimulated osteogenic differentiation of rat BMSCs was increased by overexpression of Wnt3a, while the opposite was observed in the Wnt3a-deficient cells. Moreover, the decreased osteogenic differentiation capacity by increased expression of miR-195-5p was rescued by Wnt3a overexpression, showing miR-195-5p directly targeted Wnt3a.