MicroRNA-495 downregulates AQP1 and facilitates proliferation and differentiation of osteoblasts in mice with tibial fracture through activation of p38 MAPK signaling pathway

Lei Zhu,Zun-Wen Lin,Gang Wang,Hong Zhang,Qing-Jia Xu,Ben Liu

doi:10.1038/s41598-019-50013-6

Abstract

Osteoblasts are implicated in the building of the vertebrate skeleton. The current study aimed to investigate the role of microRNA-495 (miR-495) in the osteoblasts of mice with tibial fractures and the underlying mechanism involving in aquaporin-1 (AQP1) and the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. Initially, a microarray-based analysis was performed to screen the differentially expressed genes and miRNAs associated with tibial fracture. Following the establishment of a tibial fracture mouse model, the positive rate of the AQP1 protein in the fracture tissue was detected by immunohistochemistry (IHC). Next, to verify the binding site between miR-495 on AQP1, bioinformatics data were employed in addition to the application of a dual-luciferase reporter gene assay. The osteoblast cell line MC3T3-E1 was treated with miR-495 mimic, miR-495 inhibitor and Anisomycin to explore the potent effects of miR-495 on proliferation and differentiation of osteoblasts in mice with tibial fracture. The expression of miR-495, AQP1, p38 MAPK, PCNA, Cyclin D1, OCN, and OPN was subsequently evaluated by RT-qPCR and Western blot analysis. Cell viability, the number of calcium nodules and alkaline phosphatase (ALP) activity were detected by MTT assay, alizarin red staining, and ALP activity assay, respectively. Our results revealed that miR-495 was down-regulated while AQP1 was up-regulated in the mice with tibial fractures. AQP1 was verified as a target gene of miR-495. When the cells were treated with overexpressed miR-495 or activated p38 MAPK signaling pathway, elevated expression of PCNA, Cyclin, D1, OCN, and OPN along with an increased amount of calcium nodules, higher cell viability, and enhanced ALP activity was detected, while the expression of AQP1 was reduced. Collectively, the key findings of the present study support the notion that overexpressed miR-495 may activate the p38 MAPK signaling pathway to inhibit AQP1 and to promote the proliferation and differentiation of osteoblasts in mice with tibial fracture.

Highlights

Tibia fracture is an injury often accompanied by long periods of immobility and disuse which may lead to a permanent reduction in bone mineral density (BMD), predisposing an individual to osteoporotic fractures in later life[1]
The results indicated that miR-495 could potentially influence fracture healing via its regulation on AQP1 and can be regarded as a promising target for fracture treatment (Fig. 1C)
The amount of calcium nodules in the miR-495 inhibitor group was notably lower than that in the blank group (p < 0.05). All these results indicate that the over-expression of miR-495 or activation of the p38 MAPK signaling pathway accelerates the formation of calcium nodule of osteoblast MC3T3-E1 in fracture mice

Summary

Introduction

Tibia fracture is an injury often accompanied by long periods of immobility and disuse which may lead to a permanent reduction in bone mineral density (BMD), predisposing an individual to osteoporotic fractures in later life[1]. The coordinative role of various types of cell types has been linked to the process of bone formation, remodeling and healing[2]. The formation of bone is a highly regulated and intricate developmental process, which relates to the differentiation of mesenchymal stem cells into osteoblasts[3]. Accumulating studies have highlighted the role of microRNAs (miRNAs) in osteoblast differentiation and bone formation, such as miR-2861 and miR-142-3p9,10. A study has indicated the role of the p38 MAPK signaling pathway in the down-regulation of AQP113. We hypothesized that miR-495 may have effects on the osteoblast proliferation and differentiation through the p38 MAPK signaling pathway. During the current study we aimed to investigate the capacity of miR-495 to promote the proliferation and differentiation of osteoblasts in mice with tibial fracture through the p38 MAPK signaling pathway

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Discussion

Conclusion