MiR-129 Targeting Smad3 Mediate IHH Signaling Pathway To Regulate Acetabular Roof Cartilage Ossification Delayed In DDH

Background and objective:Developmental dysplasia of the hip(DDH)is one of the most complex three-dimensional morphological deformities in the immature hips,with high disability rate and unclear etiology.Previous DDH etiology studies have focused on Y-type cartilage.Recently,our 3D-CT and MRI studies have confirmed cartilage ossification abnormalities in the acetabular roof of children with DDH,but the specific mechanism remains unclear.microRNA have been confirmed plays an important role in the regulation of chondrocyte proliferation,differentiation and cartilage ossification,but its research on DDH has not been reported.This study intends to establish DDH animal models by simulating swaddling position,expressing the microarray to analyze the abnormal expression of microRNA in the acetabular roof cartilage,predict its target gene to demonstrate that microRNA and its target gene can regulate acetabular roof cartilage ossification delayed in DDH.Methods:(1)4-week old New Zealand rabbits were utilized to establish DDH models,making left knee joints straightened and fixed with long leg cylinder cast to simulate straight-leg swaddling position.The contralateral hip joint was utilized as control group.All rabbit models were examined by X-ray;(2)The abnormal changes of acetabulum were detected by X-ray,MRI,tissue specimen and Safranin O-fast green staining;the organelle structure was detected by transmission electron microscopy;(3)Differential expression of miRNAs in acetabular roof cartilage samples of the model group and the control group were analyzed,and screening of miRNAs closely related to cartilage development for verification;Detected the existence miRNA in acetabular cartilage by in situ hybridization;(4)Rabbit acetabular chondrocytes were isolated and cultured,the second generation of cultured chondrocytes were identified by type II collagen immunofluorescence staining;(5)Luciferase expression assay to detect the targeted relationship between the selected miRNA and downstream genes;(6)Down-regulated the selected miRNA,and the expression changes of downstream target gene were detected by PCR and Western blot and proliferation of chondrocytes was detected by CCK-8.ELISA was used to detect the secretion of type ? collagen and proteoglycan of chondrocytes.Results:(1)13 DDH rabbit models were successfully established.The MRI showed that the acetabular apical cartilage showed ossification abnormality.The reddish O-fast green staining showed that the model group was thicker than the control group.Transmission electron microscopy showed that the chondrocytes of the model group were broken and the organelles were reduced;(2)The miRNA microarray result showed the expression of miR-129 was lower in model group and the result was verified.The in situ hybridization showed miR-129 existed in acetabular cartilage;(3)Chondrocytes grew with adherence and the shape of cells was triangle or polygonal mostly.After immunofluorescence staining for Col?,the cytoplasm of chondrocytes turn to red,and the red particles are found around the nucleus;(4)Luciferase expression assay confirmed that miR-129 targets Smad3;PCR and Western blot confirmed that inhibited miR-129 increased Smad3,down-regulated IHH caused cartilage ossification delayed,and CCK8 showed chondrocytes proliferation decreased significantly.The ELISA showed that the secretion of PG in chondrocytes was reduced,and there was no significant change in Col?,TUNEL showed chondrocytes apoptotic rate increased.Conclusion:The research confirmed for the first time that miR-129 is expressed in chondrocytes,and down-regulated miR-129 targeting to increase Smad3,resulting in low expression of IHH,suppressing proliferation and differentiation of chondrocytes,and chondrocytes ossification delayed.miR-129 is expected to be a molecular marker and a new therapeutic target for early diagnosis of DDH.