Abstract
BackgroundMechanical loading is an essential factor for bone formation. A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation.MethodsMouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro. Using molecular and biochemical methods, IGF-1 (insulin-like growth factor-1), PGE2 (prostaglandin E2), OPG (osteoprotegerin) and NOS (nitric oxide synthase) activities of the cells were assayed. MiR microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. MC3T3-E1 osteoblasts were stimulated by the mechanical tensile strain, and the miR-29b-3p expression was detected with miR microarray and RT-qPCR. Additionally, the effect of miR-29b-3p on IFG-1 secretion of osteocytes and the influence of conditioned medium of osteocytes transfected with miR-29b-3p on osteoblast differentiation were investigated.ResultsThe mechanical strain increased secretions of IGF-1 and PGE2, elevated OPG expression and NOS activities, and resulted in altered expression of the ten miRs, and possible target genes for these differentially expressed miRs were revealed through bioinformatics. Among the ten miRs, miR-29b-3p were down-regulated, and miR-29b-3p overexpression decreased the IGF-1 secretion of osteocytes. The mechanical strain did not change expression of osteoblasts’ miR-29b-3p. In addition, the conditioned medium of mechanically strained osteocytes promoted osteoblast differentiation, and the conditioned medium of osteocytes transfected with miR-29b-3p mimic inhibited osteoblast differentiation.ConclusionsIn osteocytes (but not osteoblasts), miR-29b-3p was responsive to the mechanical tensile strain and regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically strained osteocytes.
Highlights
Mechanical stimulation plays an essential role in the metabolic balance of bone
We found that mechanical tensile strain down-regulated miR-29b-3p expression of MLO-Y4 cells, not MC3T3-E1 cells, and miR-29b-3p inhibited Insulin-like growth factor-1 (IGF-1) secretion of osteocytes (Fig. 3c)
Following osteocytes’ response to this mechanical loading, 10 mechanoresponsive miRs in MLO-Y4 osteocytes were identified, bioinformatics analysis revealed the possible target genes of these miRs, and 29 target genes were correlated with bone formation
Summary
As the dominant cells in bone tissue, osteocytes respond to mechanical stimulation, sense and integrate mechanical stimuli into biochemical signals to regulate both bone formation and resorption [5]. Our previous study confirmed that a mechanical tensile strain of 2500 με at 0.5 Hz for 8 h promoted osteogenesis and mechanoresponsive miRs in osteoblasts were identified [14]. A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. In osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation
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