Downregulation of miR-21 promotes tibial fracture healing in rabbits through activating ERK pathway

J. Sheng, W.-D. Liang, C.-H. Xun, T. Xu, J. Zhang, W.-B. Sheng

Department of Spine Surgery, the First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang Uyghur Autonomous Region, China. wbsheng@vip.sina.com

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• Orthopedics

OBJECTIVE: The aim of this study was to investigate the effect of micro ribonucleic acid (miR)-21 on tibial fracture healing in rabbits by regulating the extracellular signal-regulated kinase (ERK) signaling pathway, and to explore its possible underlying mechanism.

MATERIALS AND METHODS: A total of 15 healthy male rabbits were randomly divided into three groups, including: model group A (fracture group, n=5), model group B (fracture treatment group, n=5), and model group C (miR-21 siRNA + treatment group, n=5). Fracture healing was observed by imaging. The content of the serum collagen I and collagen II in rabbits was detected via enzyme-linked immunosorbent assay (ELISA). The morphology of bone tissues was observed via staining. Moreover, the expressions of ERK, transforming growth factor-β1 (TGF-β1), and Smad in osteoblasts of tibia were observed via Western blotting and Reverse Transcription-Polymerase Chain Reaction (RT-PCR), respectively.

RESULTS: There was bony callus formation in group B and C when compared with group A. Compared with group B, bony callus formation was significantly accelerated in group C, while healing cycle was shortened. Hematoxylin-eosin (HE) staining and Masson staining indicated that compared with group A, group C had more fibrous calluses, new capillaries, and fibroblasts in tissues. Meanwhile, group C exerted better maturity of collagen tissues and higher osteoid content at 20 d after modeling. Compared with group C, there were more osteoid tissues with poor maturity in group B. Meanwhile, intramembranous bone formation was deformed, and collagen content was remarkably lower in group B. The content of serum collagen I and collagen II remarkably increased in group B compared with group A (p<0.05). However, it was significantly upregulated in group C compared with group B, showing statistically significant differences (p<0.05). According to the results of Western blotting, the protein expressions of TGF-β1, Smad, and ERK in osteoblasts were significantly upregulated in group B when compared with those in group A (p<0.05). However, they increased remarkably in group C when compared with group B (p<0.05). Besides, RT-PCR results revealed that the messenger RNA (mRNA) expressions of TGF-β1, Smad, and ERK in osteoblasts were significantly higher in group B than those in group A (p<0.05). However, they were markedly raised in group C in comparison with group B (p<0.05).

CONCLUSIONS: Down-regulation of miR-21 promotes tibial fracture healing in rabbits by activating the ERK signaling pathway.

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