Abstract
BackgroundOsteoporosis seriously disturbs the life of people. Meanwhile, inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. It was reported that miR-27a-3p reduced the symptoms of osteoporosis. However, the mechanism by which miR-27a-3p in osteogenic differentiation remains largely unknown.MethodsTo induce the osteogenic differentiation in MC3T3-E1 cells, cells were treated with osteogenic induction medium (OIM). RT-qPCR was used to evaluate the mRNA expression of miR-27a-3p and CRY2 in cells. The protein levels of CRY2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and the phosphorylation level of extracellular regulated protein kinases (ERK) 1/2 in MC3T3-E1 cells were evaluated by western blotting. Meanwhile, calcium nodules and ALP activity were tested by alizarin red staining and ALP kit, respectively. Luciferase reporter gene assay was used to analyze the correlation between CRY2 and miR-27a-3p.ResultsThe expression of miR-27a-3p and the phosphorylation level of ERK1/2 were increased by OIM in MC3T3-E1 cells, while CRY2 expression was decreased. In addition, OIM-induced increase of calcified nodules, ALP content and osteogenesis-related protein expression was significantly reversed by downregulation of miR-27a-3p and overexpression of CRY2. In addition, miR-27a-3p directly targeted CRY2 and negatively regulated CRY2. Meanwhile, the inhibitory effect of miR-27a-3p inhibitor on osteogenic differentiation was reversed by knockdown of CRY2 or using honokiol (ERK1/2 signal activator). Furthermore, miR-27a-3p significantly inhibited the apoptosis of MC3T3-E1 cells treated by OIM. Taken together, miR-27a-3p/CRY2/ERK axis plays an important role in osteoblast differentiation.ConclusionsMiR-27a-3p promoted osteoblast differentiation via mediation of CRY2/ERK1/2 axis. Thereby, miR-27a-3p might serve as a new target for the treatment of osteoporosis.
Highlights
Osteoporosis seriously disturbs the life of people
All these results indicated that miR-27a-3p and p-ERK1/2 expression was upregulated in ODMtreated MC3T3-E1 cells, while CRY2 was inactivated
The expressions of osteoblast differentiation-related proteins (OCN, OPN and Runx2) and p-ERK1/2 in MC3T3-E1 cells were notably decreased by CRY2 overexpression or miR27a-3p inhibitor (Fig. 2e). These results revealed that miR-27a-3p inhibitor or CRY2 overexpression could inhibit the osteoblast differentiation in MC3T3-E1 cells through inactivation of ERK1/2 signaling pathway
Summary
Osteoporosis seriously disturbs the life of people. inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. Silencing of CRY2 accelerated the acetylation of histone 3 by activating the CLOCK/BMAL1/P300 signaling pathway, and promoted the osteogenic bone formation and cell autophagy through promoting a transcription complex with Runt-related transcription factor 2 (Runx2) (Tang et al 2020). Honokiol could induce the activation of ERK signaling pathway (Yeh et al 2016). Honokiol and p-ERK promoted osteoblast differentiation (Boot et al 2016; Yeh et al 2016). Multiple reports indicated that ERK1/2 pathway could promote osteoblast differentiation and bone formation (Kim et al 2019). Puerarin and astragalin promoted osteogenic differentiation in MC3T3 and MG-63 cells by activating ERK1/2 to prevent osteoporosis (Liu et al 2019; Wang et al 2013). The relationship between these two ingredients in osteoblast differentiation has not been reported
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