MiR-140-3p overexpression activates the Wnt signaling pathway to promote fracture healing

Q.-P. Liu, T.-H. Wu, H. Zheng, S.-J. Chen, Y.-H. Chen, L. Chen, Z. Lin

Department of Orthopedics, Mindong Hospital Affiliated to Fujian Medical University, Fu’an, China. lin87zhen@163.com

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• Orthopedics

OBJECTIVE: To study the mechanism of micro ribonucleic acid (miR)-140-3p participating in the regulation of fracture healing in rats.

MATERIALS AND METHODS: A total of 50 male Sprague-Dawley (SD) rats were randomly divided into five groups, namely, group A [phosphate-buffered saline (PBS)] (n=10), group B (miR-140-3p mimics) (n=10), group C [ mimics negative control (NC)] (n=10), group D [antisense oligonucleotide (ASO)-miR-140-3p] (n=10), and group E (ASO NC) (n=10). A rat model of fracture was established on all the rats through the operation. From the successful establishment of the model, the rats in group A were intraperitoneally injected with 50 μL PBS (2 nmol) once a week for 6 weeks, and those in group B, C, D, and E were injected with equivalent volume of miR-140-3p mimics, mimics NC, ASO-miR-140-3p, and ASO NC, respectively, once a week since the successful establishment of model for 6 weeks. The fracture healing in the rats was evaluated via imaging. Meanwhile, Real Time-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression of miR-140-3p in the five groups. Wnt and β-catenin expressions in the five groups were detected by means of Western blotting (WB). Alkaline phosphatase (ALP) and its quantized statistical value in the five groups were detected through immunohistochemical staining.

RESULTS: The expression of miR-140-3p was stimulated in miR-140-3p mimics group and inhibited in ASO-miR-140-3p group. The detection of the miR-140-3p expression level in the five groups via RT-PCR showed that miR-140-3p mimics group had a remarkably higher miR-140-3p expression than the other four groups. The differences were statistically significant (p<0.05). The WB assay verified that the Wnt and β-catenin expressions in miR-140-3p mimics group were notably higher than those in control groups, and there were statistically significant differences (p<0.05). Compared with those in the groups injected with PBS, ASO miR-140-3p, mimics NC, and ASO NC, there were evidently more callus tissues, better healed and more blurred fracture lines, as well as no translocation and looseness of internal fixation, in the group injected with miR-140-3p mimics, suggesting that the stimulation of the miR-140-3p expression promotes the fracture healing in the rats. The results of immunohistochemical staining indicated that the number of ALP-positive osteoblasts in the rats in miR-140-3p mimics group was increased markedly in comparison with that in the remaining groups (p<0.05), implying that the differentiation of osteoblasts in the rats was affected in miR-140-3p mimics group.

CONCLUSIONS: The overexpressed miR-140-3p in the rats with fracture can promote fracture healing by activating the Wnt signaling pathway.

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