REMOVED: MiR-539-3p inhibits osteoblast activity and bone formation in vivo via targeting LRP-6

Abstract

X-linked hypophosphatemia (XLH), the most common form of inherited rickets, is characterized by renal phosphate (Pi) wasting, abnormal vitamin D metabolism and defective bone mineralization. It is caused by an inactivation mutation in PHEX gene. Other factors, such as microRNA (miRNA), which regulates biological functions by interfering with specific mRNA translation, also contribute in controlling disease pathogenesis. Recent studies indicate the role of miRNAs in osteoblast functions. The main objective of this study is to identify novel miRNAs, regulated by Phex in osteoblast cells, and to determine whether miR-539-3p, a known tumor suppressor, aids in osteogenesis. MiRNA expression patterns in control and siPhex treated cells were analyzed by miRNA microarray profiling and quantitative RT-PCR. Effect of miR-539-3p on osteoblast differentiation was achieved by transfection with miR-539-3p and anti-miR in mouse calvarial osteoblasts. MiR-539-3p targets were identified by Luciferase reporter gene assay. LRP-6, Wnt3, Beta catenin, p-beta catenin, GSK3β and Lef-1 protein levels were determined by Western blotting. To investigate the function of miR-539-3p in vivo, animal groups were divided into; sham+PBS (ovary intact), ovariectomized (Ovx)+miC, Ovx+miR and Ovx+anti-miR. Chemically modified oligonucleotide miR and anti-miR were injected at 5ug/animal dose via a subcutaneously injection into mice. In PHEX silenced mice calvarial osteoblast cells, miR-539-3p was up-regulated as analyzed by miRNA array profiling and qRT-PCR analysis. Over-expression of miR-539-3p inhibited osteoblast differentiation, whereas inhibition of miR-539-3p function promoted expression of osteoblast-specific genes and alkaline phosphatase (ALP) activity. Target prediction analysis tools and experimental validation by luciferase 3ⲠUTR reporter assay identified LRP-6 as a direct target of miR-539-3p. Over-expression of miR-539-3p led to LRP-6 repression and inhibition of Wnt-Beta catenin signaling. Furthermore, miR-539-3p silencing led to increased bone formation and improved trabecular micro architecture in Ovx mice. Although miR-539-3p is known to be a tumor repressor, we identified a second complementary function of miR-539-3p where it inhibits LRP-6-mediated osteogenesis. Our findings suggest that pharmacological inhibition of miR-539-3p by anti-miR-539-3p could represent a therapeutic strategy for enhancing bone formation in vivo.