Background  microRNA(miR) is a non-encoded small molecule compound that plays an important role in regulation of biological functions (such as osteogenesis).

  Objective  To investigate the effect of miR-129-5p on osteogenic differentiation of preosteoblast cell line (MC3T3-E1) and its related mechanisms.

  Methods  MC3T3-E1 cells were cultured in vitro and transfected with lentiviral vectors (miR-control, miR-129-5p mimic and miR-129-5p inhibitor) to overexpress or knock down miR-129-5p levels. After osteogenic induction of cultured cells, alkaline phosphatase (ALP) staining kit was used to detect the activity of alkaline phosphatase, and alizarin red staining (ARS) staining was used to observe the formation of calcium nodules to detect the level of osteogenic differentiation. The mRNA and protein expression levels of Runx2 and OCN genes were tested by real-time fluorescent quantitative PCR (qRT-PCR) and western blot methods. Bioinformatics methods were used to predict the target gene of miR-129-5p. The luciferase reporter gene assay was performed to confirm the binding relationship with miR-129-5p.

  Results  MC3T3-E1 cells were transduced with lentiviral vectors (miR-control, miR-129-5p mimic and miR-129-5p inhibitor) for osteogenic differentiation. Compared with the control group, MC3T3-E1 cells transduced with miR-129-5p mimic showed increased alkaline phosphatase staining and expression of early osteogenic marker Runx2 after 7 days. The level of late osteogenic marker OCN in mimic group was up-regulated after 14 days. ARS results showed larger and more calcified nodules than control group after 21 days (P＜0.05). In the miR-129-5p inhibitor group, ALP activity was reduced and calcium nodules were smaller and fewer, and the expression of osteogenic markers Runx2 and OCN were down-regulated (P＜0.05). The target gene of miR-129-5p was predicted to be Smad6 via Targetscan website and dual luciferase report verified the relationship. After overexpression of miR-129-5p, the expression level of Smad6 protein was repressed.

  Conclusion  miR-129-5p may promote osteogenic differentiation of MC3T3-E1 cells by targeting Smad6.