miR-138-5p inhibits osteoblast differentiation by downregulating SIRT1 expression under simulated weightlessness

  Background  Mechanical stimulation plays a vital role in maintaining bone remodeling and homeostasis, and mechanical unloading-induced skeletal system damage is one of the main limitations for long-term space flight. miRNA and SIRT1 can regulate osteoblasts functions via regulating signaling pathways related to bone formation.

  Objective  To explore the effect of miR-138-5p/SIRT1 signaling pathway on osteoblast differentiation under simulated weightlessness condition, and provide a therapeutic target for intervention of weightlessness-induced bone loss.

  Methods  2D clinostat was used to culture MC3T3-E1 under simulated weightlessness to verify the changes of expression of SIRT1. miR-138-5p mimic, miR-138-5p inhibitor, pcDNA3.1(+)-SIRT1 and the corresponding negative control were transfected into MC3T3-E1 cells for functional experiments, MC3T3-E1 cells were treated with simulated weightlessness for 48h after transfecting with inhibitor-NC or miR-138-5p inhibitor, and qRT-PCR was used to detect the mRNA expressions of miRNA and SIRT1, Runx2, Osx mRNA. The protein expression of SIRT1, Runx2 and Osx were detected by Western blot, and Alkaline phosphatase (ALP) activity and ALP staining were used to detect osteoblast differentiation.

  Results  The expressions of Runx2, SIRT1 mRNA and protein in MC3T3-E1 cells decreased under simulated weightlessness (P＜0.01 or P＜0.05); After transfecting miR-138-5p mimic or inhibitor into MC3T3-E1 cells, qRT-PCR, Western Blot, ALP activity and ALP staining detection showed that miR-138-5p significantly inhibited osteoblast differentiation (P＜0.01 or P＜0.05). After miR-138-5p mimic and pEX-SIRT1 being co-transfecting into MC3T3-E1 cells, the result showed that miR-138-5p negatively regulated osteoblast differentiation via inhibiting SIRT1 (P＜0.01). Furthermore, knockdown of endogenous miR-138-5p could alleviate the suppression of SIRT1 expression and osteoblastic differentiation under simulated weightlessness (P＜0.01).

  Conclusion  SIRT1 is downregulated while miR-138-5p is upregulated under simulated weightlessness. miR-138-5p regulates the protein expression level of Runx2 and Osx via targeting SIRT1 and inhibits osteoblast differentiation. In addition, silencing miR-138-5p/SIRT1 alleviates the suppression effect of simulated weightlessness on osteoblast differentiation, indicating miR-138-5p may be a potential target for weightlessness-induced bone loss.