MiR-374b promotes osteogenic differentiation of MSCs by degrading PTEN and promoting fracture healing

J.-B. Ge, J.-T. Lin, H.-Y. Hong, Y.-J. Sun, Y. Li, C.-M. Zhang

Department of Traumatic Orthopedics, Yantaishan Hospital, Yantai, China. zhangchunmingjn@outlook.com

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• Orthopedics

OBJECTIVE: To investigate whether miR-374b can promote the differentiation of MSCs into osteoblasts by mediating PTEN, thus promoting fracture healing.

MATERIALS AND METHODS: Primary cultured mouse mesenchymal stem cells were obtained for following experiments. Flow cytometry was used to determine the expression of MSCs surface antigens to identify the purity. Alizarin red staining was used to detect whether MSCs could differentiate into osteoblasts. QRT-PCR was used to detect the expression of osteogenic marker genes as well as miR-374b and PTEN in bone marrow-derived MSCs from fractured mice model. ALP activity detection kit was used to detect ALP activity in cells. Changes of osteogenic proteins in cells were evaluated by Western blot. Bioinformatics methods were used to predict the binding sites between miR-374 and target genes. Luciferase reporter assay was used to confirm whether miR-374 could bind to the target gene.

RESULTS: Under normal culture, MSCs grew into a long fusiform shape on the 4th day. After induced in the osteogenic induction medium for seven days, calcified nodules appeared. The results of the detection of MSCs surface antigen markers showed that CD90 was 99.12%, and CD45 was 0.23%. Alizarin red staining showed that MSCs possess the ability to differentiate into osteogenic. The expression level of MiR-374b and PTEN increased significantly in the early stage of fracture in mice, but no significant difference was observed at a later stage. After overexpression of miR-374b, the cell ALP activity, the expression of osteogenesis-related genes and osteogenesis-related proteins was significantly increased, while after knocking out miR-374b, the opposite result was observed. The result of luciferase reporting assay showed that miR-374b can bind to PTEN. As mentioned above, overexpression of miR-374b resulted in upregulation of osteogenic-related genes and proteins, while over-expression of both PTEN and miR-374b could partly reverse the outcomes.

CONCLUSIONS: miR-374b can promote osteogenic differentiation of MSCs by degrading PTEN, thereby promoting fracture healing.

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