Molecular Biological Mechanism Of MiR-1-3p In Abnormal Ossification Of The Acetabular Roof Cartilage In DDH

BackgroundDevelopmental dysplasia of the hip(DDH)is a highly prevalent hip disease among children.However,its pathogenesis remains unclear.MicroRNAs(miRNA)are important regulators of cartilage development.In a previous study,high-throughput miRNA sequencing of tissue samples from an animal model of DDH showed a low level of miR-1-3p in the cartilage of the acetabular roof(ARC),but its role in DDH pathogenesis was not addressed.Therefore,our aim here was to investigate the effects of miR-1-3p in the ARC MethodsThe diagnosis of acetabular dysplasia was confirmed with X-ray examination,while imaging and HE staining were conducted to further evaluate the ARC thickness in each animal model.FISH was employed to verify miR-1-3p expression in the ARC and chondrocytes.The miR-1-3p target genes were predicted by a bioinformatics database.A dual-luciferase reporter 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 was evaluated by qPCR analysis.The protein expression of SOX9,RUNX2 and collagen type X was detected by western blot analysis.The levels of SOX9,RUNX2,and collagen type X in the ARC were further assessed via immunohistochemistry analysis.Finally,Alizarin Red S staining was used to observe the mineralized nodules produced by the chondrocytes.ResultsWe observed low expression of miR-1-3p in the ARC of animals with DDH.SOX9 is a miR-1-3p target gene.Using miR-1-3p silencing technology in vitro,we demonstrated significantly reduced chondrocyte-generated mineralized nodules compared to those of the control.We also confirmed that with miR-1-3p silencing,SOX9 expression was upregulated,whereas the expression of genes associated with endochondral osteogenesis such as RUNX2 and collagen type X was downregulated.To confirm the involvement of miR-1-3p silencing in abnormal ossification through SOX9,we also performed a rescue experiment in which SOX9 silencing restored the low expression of RUNX2 and collagen type X produced by downregulated miR-1-3p expression.Finally,the elevated SOX9 levels and reduced RUNX2 and collagen type X levels in the ARC of rabbits with DDH were also verified using immunohistochemistry,RT-PCR,and western blots.ConclusionThe relatively low expression of miR-1-3p in the ARC may be the cause of abnormal endochondral ossification in the acetabular roof of animals with DDH.