MiR-25 overexpression promotes fracture healing by activating the Wnt signaling pathway

Y. Lang, Q. Sun, L.-M. Zhu, X.-D. Qiu, B.-S. Hu, J. Yang, J.-D. Zhang

Department of Orthopaedic Surgery, Fuyang Orthopaedics and Traumatology Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China. zhang_jd@163.com

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• Orthopedics

OBJECTIVE: To study the mechanism of micro-ribonucleic acid (miR)-25 in regulating the fracture healing in rats.

MATERIALS AND METHODS: A total of 45 male Sprague-Dawley (SD) rats were selected and randomly divided into group A [Phosphate Buffered Saline (PBS), n=15], group B (mimics NC, n=15) and group C (miR-25 mimics, n=15). The fracture model in rats was established via operation in all groups. From 1 d after the successful modeling, 50 μL (2 nmoL) of PBS was intraperitoneally injected into rats in group A, an equal amount of mimics NC was injected into rats in group B, and an equal amount of miR-25 mimics was injected into rats in group C. The above agents were injected once a week for consecutive 6 weeks. Fracture healing in rats was evaluated via X-ray imaging. At the same time, miR-25 expression in the three groups was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Protein expressions of β-catenin, proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) in the three groups were detected via Western blotting. The OCN-, PCNA- and BMP-2-positive osteoblasts in the three groups were detected via immunohistochemical staining and were further quantified. Moreover, the biomechanical properties of femoral fracture healing in the three groups were analyzed via the 4-point bending flexural test.

RESULTS: The X-ray examination of the femoral fracture healing at postoperative 1 and 7 weeks revealed that the fracture line disappeared, and both callus formation and fracture healing were good in miR-25 mimics group. In PBS group and mimics NC group, a few fracture lines could be observed, and both callus formation and fracture healing were poor. RT-PCR data showed that the expression level of miR-25 significantly increased in the miR-25 mimics group compared with that in the other two groups, and the differences were statistically significant (p<0.01). Western blotting analyses showed upregulated levels of β-catenin, PCNA and BMP-2 in the miR-25 mimics group compared with those in the control group, and the differences were statistically significant (p<0.01). Immunohistochemical staining manifested that the numbers of OCN-, PCNA- and BMP-2-positive osteoblasts in miR-25 mimics group markedly increased compared with that in the other two groups (p<0.01), suggesting that osteoblast differentiation in miR-25 mimics group was affected. The above immunohistochemical results indicated that the osteoblast differentiation at the fracture end in miR-25 mimics group was markedly enhanced compared with that in control groups. The results of the biomechanical test of femur specimens at 7 weeks after operation showed that in miR-25 mimics group, the maximum load, fracture energy and stiffness increased by 188%, 333% and 90%, respectively, compared with those in the PBS group (p<0.01). It is indicated that miR-25 promoted the mechanical properties of fracture healing.

CONCLUSIONS: The overexpression of miR-25 in the fracture in rats promotes fracture healing by activating the Wnt signaling pathway.

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