Abstract
MicroRNAs (miRNAs) play a key role in the regulation of almost all the physiological and pathological processes, including bone metabolism. Recent studies have suggested that miR-27 might play a key role in osteoblast differentiation and bone formation. Increasing evidence indicates that the canonical Wnt signaling pathway contributes to different stages of bone formation. In this study, we identify miR-27a can promote osteoblast differentiation by repressing a new target, secreted frizzled-related proteins 1 (sFRP1) expression at the transcriptional level. Here, 21 candidate targets of miR-27a involved in canonical Wnt/β-catenin signaling were predicted, and a significant decrease in sFRP1 luciferase activity was observed both in 293T and MG63 cells co-transfected with the matched luciferase reporter constructs and miR-27a mimic. Furthermore, the presence of exogenous miR-27a significantly decreased sFRP1 mRNA and protein expression in hFOB1.19 cells during both proliferation and osteogenic differentiation. The over-expression of miR-27a or knockdown sFRP1 significantly increased the percentage of apoptotic hFOBs, the percentage of cells in the G2-M phase of the cell cycle and the expression of key osteoblastic markers, including ALP, SPP1, RUNX2 and ALP activity. Over-expression of miR-27a or knockdown of endogenous sFRP1 led to an accumulation of β-catenin in hFOBs. In the present study, we demonstrate that miR-27a induced gene silencing effect is a vital mechanism contributing to bone metabolism in hFOB cells in vitro, which is partly affected by the post-transcriptional regulation of sFRP1, during osteoblast proliferation, apoptosis and differentiation.
Highlights
Increasing evidence indicates that Wnt signaling plays a critical role in the development and maintenance of many organs and tissues, including bone [1,2]
The findings indicate that the ideal proliferation in vitro for human fetal osteoblastic 1.19 cell line (hFOB) occurs in the non-differentiation medium at 33.4uC and the optimal osteogenesis occurs in the osteogenic medium at 39.4uC, as previously shown [27,28,33,34,35,36]
MiR27a expression was higher in MG63 cells and was the highest in miR-27a inhibitor negative control (NC) for siR secreted frizzled-related proteins 1 (sFRP1)+ miR-27a inhibitor
Summary
Increasing evidence indicates that Wnt signaling plays a critical role in the development and maintenance of many organs and tissues, including bone [1,2]. The regulation of bone metabolism via the canonical Wnt signaling pathway has recently become a focus of research [4,5]. Wnts binding to frizzled and LRP5/6 co-receptors activate canonical signaling pathways [2,6]. Secreted frizzled-related proteins (sFRPs) contain a domain similar to frizzled proteins, which binds directly to Wnts as endogenous Wnt antagonists [7,8]. The recent findings demonstrate that sFRPs can either promote or suppress Wnt/betacatenin signaling. The promotion or suppression depends on cellular context, concentration and the expression pattern of frizzled receptors [9]
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