Abstract:
Background MicroRNAs (miRNA) are endogenous non-coding RNAs holding a nonnegligible position in every aspects of life, especially in bone remodeling. Among these microRNAs, the interaction between miR-134-5p and its potential target gene Integrin β1(Itgb1) is still unknown in osteoclastogenesis.
Objective To study the effects of miR-134-5p on osteoclastogenesis via targeting Itgb1.
Methods The osteoclastic differentiation of mouse bone marrow-derived macrophages (BMMs) was induced into osteoclasts and the morphology of osteoclasts was observed by TRAP staining. The expression levels of osteoclast-related genes TRAP, CTSK and NFATc1 as well as miR-134-5p were detected by qRT-PCR. The experiment included four groups, miR-134-5p overexpression (agomir) group, downregulating miR-134-5p (antagomir) group, and the control groups including agomir NC group and antagomir NC group, then the cells were transfected into BMMs. qRT-PCR was then utilized to determine the transfection efficiency and the morphology of osteoclasts was observed by TRAP staining and F-actin staining. The expression levels of TRAP, CTSK and NFATc1 in each group were also detected. Bioinformatic analysis were used to screen the potential target gene of miR-134-5p, and the expression levels of Itgb1 gene and protein were detected by qRT-PCR and Western blot.
Results Compared with the BMMs induced for 1 day, the osteoclasts differentiating from BMMs that induced for 7 days represented more TRAP positive multinucleated cells, and expressed an increasing level of TRAP,CTSK and NFATc1 gene (P＜0.01), while the qRT-PCR results showed that the expression level of miR-134-5p decreased (P＜0.01). The differentiation of the BMMs in osteoclasts in vitro was inhibited after transfected with agomir, with decreasing number of TRAP positive multinucleated cells and F-actin rings, as well as lower expression levels of TRAP, CTSK and NFATc1 (P＜ 0.01). However, after transfected with antagomir, the BMMs accelerated the formation of osteoclasts, which manifested as the increasing number of TRAP positive multinucleated cells along with F-actin rings, and the increased expression levels of TRAP, CTSK and NFATc1. Bioinformatic analysis showed that the binding site between Itgb1 and miR-134-5p was existed at the 3 '- UTR end of Itgb1. qRT-PCR and Western blot showed that the expression level of Itgb1 decreased when miR-134-5p was upregulated.
Conclusion miR-134-5p plays an important role in inhibiting osteoclastogenesis by targeting Itgb1.
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