miR-29b-3p regulated osteoblast differentiation via regulating IGF-1 secretion of mechanically stimulated osteocytes

Abstract

Background: Mechanical 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.

Methods: Mouse 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.

Results: The 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.

Conclusions: In 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.

Keywords: Mechanical tensile strain; Osteoblast differentiation; Osteocyte; miRNA microarray.

Fig. 1

MLO-Y4 cells responded to a mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h. a/b After MLO-Y4 cells were stimulated by the mechanical tensile strain, the contents of IGF-1 and PGE2 in the supernatant of cell culture medium were increased (n = 5). c The mechanical tensile strain increased protein levels of OPG (osteoprotegerin) of MLO-Y4 cells (n = 6). d Following mechanical tensile strain, the activities of NOS in the cells were up-regulated (n = 5). *P < 0.05 and **P < 0.01, compared with the unstrained control group

Fig. 2

A total of forty differentially expressed miRNAs were selected by microarrays. a: up-regulated miRs, b: down-regulated miRs

Fig. 3

In MLO-Y4 osteocytes, miR-29b-3p was one of the ten mechanoresponsive miRs which were confirmed by RT-qPCR. a Based on microarray assay of miRs, the result of RT-qPCR showed that ten miRs were responsive to the mechanical tensile strain, and miR-29b-3p was one of the ten miRs (n = 5). b The miR-29b-3p mimic elevated miR-29b-3p level of osteocyte and the miR -29b-3p inhibitor decreased the miR level (n = 5). c In MC3T3-E1 osteoblastic cells, the mechanical tensile strain did not change the expression of miR-29b-3p, which was demonstrated with microarray and RT-qPCR. Contrarily, in MLO-Y4 osteocytes, the mechanical strain decreased miR-29b-3p expression (n = 5). *P < 0.05 and **P < 0.01, compared with the unstrained control group, or between the indicated groups

Fig. 4

miR-29b-3p regulated IGF-1 secretion of MLO-Y4 osteocytes, and after the osteocytes were transfected with miR-29b-3p mimic or miR-29b-3p inhibitor, the osteocytes’ conditioned culture medium influenced osteoblastic differentiation of MC3T3-E1 cells. a miR-29b-3p mimic decreased IGF-1 secretion of osteocytes, and the miR inhibitor increased the IGF-1 secretion (n = 6). The conditioned medium of osteocytes treated with miR-29b-3p mimic reduced ALP activity of osteoblasts (b), BMP-2 protein (c) and Runx 2 mRNA (d) levels of osteoblasts (n = 6). In contrast, the conditioned medium of osteocytes treated with miR-29b-3p inhibitor enhanced ALP activity of osteoblasts (b), BMP-2 protein (c) and Runx 2 mRNA (d) levels of osteoblasts (n = 6). *P < 0.05 and **P < 0.01, compared with the unstrained control group, or between the indicated groups