The effect of microRNA-608 in the dynamic balance of osteogenesis and adipogenesis differentiation of human bone marrow mesenchymal stem cells

Objective To detect the expression of miR-608 in human bone marrow mesenchymal stem cells (hBMSCs) during differentiation into osteoblasts and adipocytes, and the regulatory mechanism of hBMSCs in differentiation into osteoblasts and adipocytes. Methods Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of miR-608 during the differentiation of hBMSCs into osteoblasts and adipocytes. Cell counting kit-8 (CCK-8) and clone formation assay were used to detect the effect of miR-608 on the proliferation of hBMSCs. The target gene of miR-608 was analyzed by bioinformatics software. Double luciferase reporter gene was used to detect the binding of miR-608 to target gene. Western blotting was used to detect the expression changes of the target gene protein of miR-608. Alizarin red staining and oil red O staining were used to detect the effect of miR-608 on the differentiation of hBMSCs into osteoblasts and adipocytes. Results FQ-PCR showed that the expression of miR-608 decreased significantly on the 3rd day of differentiation from hBMSCs to osteoblasts. Compared with the control group (1.00±0.11), the expression of miR-608 in osteogenic differentiation group (0.20±0.09) was significantly lower (t=9.479, P<0.01). FQ-PCR showed that the expression of miR-608 increased on the 3rd day of differentiation from hBMSCs to adipocytes. Compared with the control group (1.00±0.09), the expression of miR-608 in osteogenic differentiation group (4.36±0.55) decreased significantly (t=10.442, P<0.05), and the difference was statistically significant. CCK-8 and clonogenic assay showed that miR-608 inhibited the proliferation of hBMSCs. Bioinformatics software analyzed the binding sites of miR-608 with related transcription factor-2 (Runx2) and SHH 3’untranslated regions (3’UTR). The double luciferase reporter gene assay showed that the luciferase activity in the group of miR-608 mimics (0.21±0.11) was significantly lower than that in the group of miR-608 mimics NC (1.00±0.22) (t=5.542, P<0.05). Western blotting analysis showed that miR-608 could inhibit the expression of Runx2 and SHH proteins. Alizarin red staining showed that the alizarin red staining was significantly lower in the miR-608 mimics group (0.32±0.12) than in the miR-608 mimics NC group (0.32±0.12) (t=6.387, P<0.05). Oil red O staining showed that the positive number of oil red O cells in the group of miR-608 mimics (32.12±6.25) was significantly higher than that in the group of miR-608 mimimics NC (19.20±2.34) (t=3.353, P<0.05). Conclusion The expression of miR-608 decreased during the differentiation of hBMSCs into osteoblasts, but increased during the differentiation of hBMSCs into adipocytes. miR-608 inhibits the proliferation of hBMSCs. miR-608 regulates the expression of Runx2 and SHH proteins by targeting Runx2 and SHH mRNA 3’UTR regions. Mi-608 inhibits the differentiation of hBMSCs into osteoblasts and promotes the differentiation of hBMSCs into adipocytes. Key words: Human bone marrow mesenchymal stem cells; Osteogenic differentiation; Adipogenic differentiation; MicroRNA