Mechanism of miR-494 Regulating Osteoblast Differentiation and Matrix Mineralization
Objective Effects of miR-494 on osteoblast differentiation and matrix mineralization and related mechanisms.Method mouse MC3T3-E1 osteoblasts were randomly divided into control group,miR-494 over-expression group and miR-494 inhibition group.The control group was not treated,and miR-494 over-expression group and miR-494 inhibition group were transfected with miR-494 MICs transfection reagent and miR-494 inhibitors transfection reagent respectively.ALP activity was detected by alkaline phosphatase(ALP)kit,and Osteocalcin(OC)activity was detected by osteocalcin radioimmunoassay kit.VonKossa staining was used to detect the number of calcified nodules in each group.The mRNA expression levels of miR-494 and phosphatase tensin homolog gene(PTEN)were detected by qRT-PCR,the protein expression levels of phosphatidylinositol 3-kinase(PI3K)/Aktpathway were detected by Western blot,and the targeting relationship between miR-494 and PTEN was verified by double Luciferase Report experiment.Result Dual luciferase experiments confirmed that PTEN was the target gene of miR-494.miR-494 mRNA and p-Akt protein in osteoblasts with miR-494 over-expression were significantly higher than those in the control group,while PTEN mRNA,ALP and OC activity and calcified nodule number were significantly lower than those in the control group;miR-494 inhibited osteoblast miR-494 mRNA and p-Akt protein were significantly lower than those in the control group and miR-494 over-expression group,while PTEN mRNA level,ALP and OC activity and calcified nodule number were significantly higher than those in the control group and miR-494 over-expression group(P<0.05).Conclusion miR-494 can effectively inhibit osteoblast differentiation and matrix mineralization,and its mechanism may be related to the regulation of Akt pathway by miR-494 mediated target gene PTEN.
NETL
NSTL
万方数据
维普
