Abstract
Dickkopf-1 (DKK1) is a powerful antagonist of canonical WNT signaling pathway, and is regarded as a biomarker for osteoporosis. Its expression is highly correlated with bone mass and osteoblasts maturation. In this study, mouse primary bone marrow cells and osteoblast cell lines were used. Luciferase reporter assay and western blotting methods were employed to validate if miRNA-433-3p epigenetically regulated DKK1 translation. Rat bone marrow derived osteoblasts were infected with lentivirus vector in which miR-433-3p was constructed. The authors constructed lentivirus mediated miRNA-433-3p stable expression and examined the alkaline phosphatase (ALP) activity and mineral deposition level in vitro. In situ hybridization method was used to observe miR-433-3p in primary osteoblasts. We built up an OVX rat model to mimic postmenopausal osteoporosis, and found aberrant circulating miR-433-3p and miR-106b, which were not reported previously. Results showed that miR-433-3p potentially regulated DKK1 mRNA, Furthermore, the correlation of serum DKK1 with circulating miR-433-3p level was significant (r = 0.7520, p = 0.046). In the luciferase reporter assay, we found that miR-433-3p siRNA decreased luminescence signal, indicating direct regulation of miR-433-3p on DKK1 mRNA. When the miR-433-3p binding site in DKK1 3’UTR was mutant, such reduction was prohibited. Western blotting result validated that miR-433-3p inhibited over 90% of DKK1 protein expression. Similarly, the change of protein expression was not observed in mutant group. The stable expression of lentivirus mediated miR-433-3p increased ALP activity and mineralization both in human and rat derived immortalized cells. We found that primary osteoblasts had higher miR-433-3p level compared with immortal cells through real-time PCR, as well as in situ hybridization experiment. Conclusively, our findings further emphasized the vital role of miR-433-3p in DKK1/WNT/β-catenin pathway through decreasing DKK1 expression and inducing osteoblasts differentiation.
Highlights
The canonical Wnt/β-catenin signaling pathway activates bone formation and resorption genes transcription
After building up the OVX rat model, selected serum miRNAs were quantified through real-time PCR method
We successfully built up an OVX rat model to mimic postmenopausal osteoporosis, and found some interesting circulating miRNAs which might played an important role in the development of osteoporosis
Summary
The canonical Wnt/β-catenin signaling pathway activates bone formation and resorption genes transcription. It is a vital factor in regulating osteoblast differentiation, proliferation, survival, and bone formation[1]. Overexpressing DKK1 in osteoblasts was found to decreased osteoblast numbers and in osteopenia[3], whereas DKK1 allele single deletion was associated with increased bone formation and bone mass in another murine model[4]. Current opinions on osteoporosis realized that DKK1 level was associated with the pathophysiology of postmenopausal osteoporosis[7, 8], and with the inflammatory cytokines effects on bone mass[9, 10]. We mainly focused on the postmenopausal osteoporosis
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