Objective To explore the influence of microRNA (miR)-27a-5p on healing of radial fractures in rats with depression.
Methods The SD rats were randomly separated into fracture control group, fracture + depression group, miR-27a-5p agomir (activator) negative control group, miR-27a-5p agomir group, miR-27a-5p antagomir negative (inhibitor) control group, and miR-27a-5p antagomir group, except for the fracture control group, the rats in the other groups were all established depression models through chronic mild and unpredictable stress stimulation. All the rats were fractured the middle part of the radius of the front right limb to prepare the fracture model, and after intervention with miR-27a-5p activator and inhibitor, the depressive symptoms of rats in each group were measured by forced swimming test and sucrose water consumption test; the amount of callus and bone microstructure of the fractured segment of the rats in each group were measured. The levels of serum inflammatory factors interleukin (IL)-2, IL-6, receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) were measured in each group; the expressions of miR-27a-5p and IL-2 mRNA in callus tissue of rats in each group were measured by real-time fluorescence quantitative (qRT-PCR) experiment. The rat bone marrow mesenchymal stem cell (BMSC) were cultured in vitro and randomly divided into control group, miR-27a-5p mimics group, miR-27a-5p mimics negative control group, and miR-27a-5p inhibitor group and miR-27a-5p inhibitor negative control group. After induced osteogenic differentiation of cells in each group and intervened in groups with miR-27a-5p mimics and inhibitors, the osteogenic differentiation of cells in each group was measured by alkaline phosphatase (ALP) staining; the expression of miR-27a-5p and IL-2 mRNA of cells in each group was measured by qRT-PCR experiment. The targeting regulation effect of miR-27a-5p on IL-2 in rat BMSC was measured by dual-luciferase reporter gene assay.
Results Compared with the fracture control group, the forced swimming immobility time was significantly prolonged, serum IL-2, IL-6 as well as RANKL levels and IL-2 mRNA expression in callus tissue were significantly increased, and the sucrose water consumption, callus volume, bone volume fraction, trabecular bone thickness, trabecular bone number, serum OPG level and expression of miR-27a-5p in bone callus tissue in the fracture + depression group were significantly decreased (P < 0.05). Compared with the fracture+depression group, the forced swimming immobility time was significantly shortened, serum IL-2, IL-6 as well as RANKL levels and IL-2 mRNA expression in callus tissue in the miR-27a-5p agomir group were significantly decreased, and the sucrose water consumption, callus volume, bone volume fraction, trabecular bone thickness, trabecular bone numbe, serum OPG level and callus tissue miR-27a-5p expression in the miR-27a-5p agomir group were significantly increased (P < 0.05); the miR-27a-5p antagomir group showed opposite trend in each index compared with the miR-27a-5p agomir group, and the difference was statistically significant (P < 0.05). Compared with the control group, the relative proportion of ALP positive cells and the expression of miR-27a-5p in the miR-27a-5p mimics group were significantly increased, and the IL-2 mRNA expression was significantly decreased (P < 0.05); the miR-27a-5p inhibitor group showed the opposite trend of cell indicators compared with the miR-27a-5p mimics group, and the difference was statistically significant (P < 0.05). The miR-27a-5p could be targeted to down-regulate IL-2 expression in rat BMSC.
Conclusion The miR-27a-5p alleviates depressive symptoms in depressed rats by reducing inflammation and promoting radial fracture healing. The molecular mechanism is that miR-27a-5p can target down-regulate IL-2 expression in rat BMSC.
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