MiR-1-3p Downregulation Delays Endochondral Ossification of the Acetabular Roof in DDH

Abstract Background: Developmental dysplasia of the hip (DDH) is a highly prevalent hip disease among children. However, its pathogenesis remains unclear. MiRNAs are important regulators in cartilage development. In previous study, miRNA high-throughput sequencing on an animal model of DDH showed a low level of miR-1-3p in the cartilage of acetabular roof (ARC), but its role in DDH pathogenesis was not addressed. Therefore, our aim was to investigate the effects of miR-1-3p in the ARC.Methods: The X-ray examination was performed to confirm acetabular dysplasia, MRI imaging and HE staining was conducted to further evaluate the ARC thickness in each animal model. The FISH was used to confirm the expression of miR-1-3p in the ARC and chondrocytes. The target gene of miR-1-3p was predicted by bioinformatics database. The dual-luciferase reporter gene assay was used to confirm the targeting relationship between miR-1-3p and SOX9. The gene expression of miR-1-3p, SOX9, RUNX2 and collagen type X by QPCR analysis. The relative proteins level was detected by western blot analysis. The level of SOX9, RUNX2 and collagen type X in the ARC through immunohistochemistry analysis. Alizarin Red S staining was used to observe the mineralized nodules produced by chondrocytes.Results: The low expression of miR-1-3p in the ARC of DDH. SOX9 is miR-1-3p target gene. Downregulation the expression of miR-1-3p in vitro and demonstrated significantly reduced chondrocytes generated mineralized nodules, as opposed to the control. We also confirmed that, with miR-1-3p silencing, SOX9 expression was upregulated, whereas expression of genes associated with endochondral osteogenesis RUNX2 and collagen type X were downregulated. To confirm the involvement of miR-1-3p silencing in abnormal ossification through SOX9, we also performed a rescue experiment where SOX9 silencing restored the low expression of RUNX2 and collagen type X, produced by miR-1-3p downregulation. Lastly, the elevated SOX9 levels and reduced RUNX2 and collagen type X levels in the ARC of DDH rabbits were also verified, using immunohistochemistry, RT-PCR, and western blot. Conclusion: The low expression of mir-1-3p in the ARC of DDH, which may be the cause of the acetabular roof abnormal endochondral ossification in DDH.