| Objective:Epiphyseal plate,also known as growth plate,is responsible for the horizontal and vertical growth of immature bones,which is a layered cartilage connecting the epiphysis and metaphysis of immature long bones.The epiphyseal plate could be injuried for many reasons,which would lead to partial or complete closure of the epiphyseal plate and would affect limb development,resulting in limb shortening and/or angular deformity.The most common cause of injury is epiphyseal fracture.10-30%of epiphyseal plate fractures would form bone bridges at the site of injury,causing premature Physeal closure(PPC),which would cause limb shortening and/or angular deformity in children,affecting children’s physical and mental health.During the inflammatory response period of growth plate injury repair,IL-1β was significantly increased.Within 24 hours after fracture injury of growth plate,the concentration of IL-1β in human synovial fluid increased by 70 times to 140 pg/mL,which increased by 7 times to 6 ng/mL.The expression of IL-1β in the growth plate during fiber formation,osteogenesis and bone bridge formation also increased to varying degrees.IL-1β disrupts the normal physiological structure of the growth plate cartilage,disrupts the growth law of the growth plate,and leads to increased proliferation of the proliferative zone,while the hypertrophy and hypertrophy of the prehypertrophic zone and the hypertrophic zone,chondrocyte apoptosis,structural disorder,and matrix structure damage,promote the ossification of the growth plate.In recent years,the research on bone bridges mainly focuses on how to treat deformities,and the research on preventing bone bridge formation is mainly through surgery.How to prevent bone bridge formation through non-invasive and effective ways is the focus of current research.The differential genes in epiphyseal chondrocytes after IL-1β simulated epiphyseal plate injury were analyzed by high-throughput sequencing of early transcriptome,bioinformatics was used to predict the potential binding sites between genes.Analysis of sequencing results showed that there were 152 differentially expressed miRNAs in epiphyseal chondrocytes after IL-1β simulated epiphyseal plate injury,of which 71 differentially expressed miRNAs were up-regulated,and 81 differentially expressed miRNAs were down-regulated,among which miR-503-5p was Significantly downregulated.MiR-503-5p,a mammalian-specific member of the miR-15/107 miRNA family,is involved in stress response,tissue differentiation and tissue remodeling.Studies have shown that high expression of miR-503-5p promotes proliferation and inhibits hypertrophic differentiation in the proliferative zone of growth plate.At the same time,studies have shown that overexpression of miR-503-5p can reduce the expression of osteogenic differentiation markers OCN,OPN and RUNX2,and reduce the number of osteoblasts.The gene prediction program(miRanda(http://www.microrna.org/microrna/home.do),TargetScan(http://www.targetscan.org),PicTar(http://pictar.mdc-berlin.de/))and target enrichment analysis was used to predict that DVL3 was a potential target gene of miR-503-5p.GO enrichment analysis,KEGG enrichment analysis and Reactome were used to analyze the miRNA signaling pathway.Wnt/β-catenin signaling pathway can regulate cartilage development and chondrocyte differentiation.β-catenin interacting protein 1(ICAT)can inhibit β-catenin in chondrocytes and reduce the proliferation area and hypertrophy area of tibial growth plate.Blocking Wnt/β-catenin signaling pathway inhibits bone formation and promotes cartilage repair at the injured growth plate.In this experiment,rat epiphyseal chondrocytes and rat bone marrow mesenchymal stem cells were cultured and rat central growth plate injury model was constructed to explore the effect and mechanism of miR-503-5p on the formation of bone bridge after epiphyseal plate injury caused by IL-1β.Methods:1.Different concentration of IL-1β(5 ng/mL,10 ng/mL,20 ng/mL and 50 ng/mL)was selected by CCK-8 and flow cytometry for subsequent experiments;2.The effect of miR-503-5p on the process of endochondral ossification of epiphyseal chondrocytes after epiphyseal injury simulated by IL-1β:(1)The effect of miR-503-5p on epiphyseal chondrocytes:Rat epiphyseal chondrocytes transfected with miR-503-5p mimic,mimic-NC,inhibitor,and inhibitor-NC,and treated with selected IL-1β for 72 hours,and then qRT-PCR and western blot were used to test the overexpression and inhibition effect of miR-503-5p,the expression of predicted potential target genes(DVL3),pathway-related genes(wntl,CTNNB1,C-myc,cyclind1,C-jun,LEF1,MET),osteogenesis-related genes(OCN,Rankl,BMP2)and cartilage-related genes(Col Ⅱa,Col X)were detected.(2)The effect of miR-503-5p on epiphyseal chondrocytes after knockdown of DVL3 expression:After epiphyseal chondrocytes were co-transfected by miR-503-5p mimic and DVL3 sirna,and treated with selected IL-1β for 72 hours.The expression of related genes was tested by qRT-PCR and Western blot.(3)The effect of miR-503-5p on epiphyseal chondrocytes was measured after the signaling pathway blocking:After epiphyseal chondrocytes were modeled and transfected with miR-503-5p,the wnt/β-catenin signaling pathway inhibitor was used to inhibit the signaling pathway.3.miR-503-5p for using IL-1β study on the effect ofbone marrow mesenchymal stem cells differentiation into bone after simulated epiphyseal plate injury:(1)The effect of miR-503-5p on bone marrow mesenchymal stem cells:use the selected concentration of IL-1β after pretreatment of rat bone marrow mesenchymal stem cells for 72 hours,transfect miR-503-5p mimic,mimic-NC,inhibitor,inhibitor-NC,and add IL-1βwith or without IL-1β after 48 hours.14 days later,alizarin red staining was carried out to analyze the calcium nodules after differentiation.qRT-PCR and Western blot were used to detect the expression of predicted potential target gene(DVL3)Expression of pathway related genes(wntl,CTNNB1,C-myc,cyclind1,C-jun,LEF1,MET),osteogenesis related genes(OCN,Rankl,BMP2)and cartilage related genes(Col Ⅱa,Col X);(2)To clarify the effect of miR-503-5p on bone marrow mesenchymal stem cells after knockdown of DVL3 expression:After modeling of bone marrow mesenchymal cells,miR-503-5p mimic and DVL3 sirna were co-transfected.Osteogenic differentiation was performed after 48 hours.Calcium nodule formation was analyzed after 2 weeks,and the differences in gene and protein expression were analyzed.(3)The effect of miR-503-5p on bone marrow mesenchymal stem cells after blocking the signaling pathway:The wnt/β-catenin signaling pathway inhibitor was used to inhibit the signaling pathway.After 48 hours,osteogenic differentiation was performed.After 2 weeks,osteogenic analysis was performed and related gene protein expression differences were detected.4.To verify the binding target of miR-503-5p and DVL3,Double luciferase assay was used.After constructing DVL3 wild-type and mutant plasmid vectors containing luciferase,miR-503-5p was co-transfected into cells.5.Animal experiment:(1)SD rats was used to establish the left epiphyseal plate central drilling injury model:The left epiphyseal plate central injury of rats was performed using a 2 mm spherical drill.To ensure the integrity of articular cartilage,the depth and angle were limited.The tissue glue was used to paste the silicon gel film at the proximal tibial cortical window,and the hole sealing was tested.The sham operation group model:2 mm spherical drill was used to drill the cortical window of the left proximal tibia without drilling the epiphyseal plate.(2)SD rats were randomly divided into sham group,NS group,IL-1β group,MI group and NC group.Drug application:drug injection was performed according to the predetermined grouping at 0d,3d,6d,9d,12d,15d and 18d respectively.NS group was given NS 20 uL,IL-1β group was given IL-1β(concentration of 10 ng/mL)20 uL,agomir and agomir-NC group were given 2nmol agomir and NC on the basis of IL-1β group.(3)Sampling:The left proximal tibia of rats was sampled at the four time points of 1,7,14,28 and 56 days after operation.(4)DR photos were taken by small animal imaging machine before sampling,and the length of bilateral tibia was compared.To analyze the proximal tibia of the injured side and to calculate TV,BV and BV/TV at each time point after epiphyseal plate injury,microCT was used.The qRT-PCR and Western blot were used to detect the expression of target DVL3.Detection of cell pathway related indicators:the expression of wntl,CTNNB1 and other signaling pathway related indicators at gene level and protein level were detected by qRT-PCR and Western blot.Osteogenic and cartilage-related indicators detection:the expression of OCN,Rankl,BMP2,Col Ⅱa and Col X at the gene level and protein level were detected by qRT-PCR and Western blot.The formation of bone bridge and its tissue morphology in each group were measured by HE staining and safranin fast green staining.Immunohistochemical staining and immunofluorescence analysis of DVL3,BMP2,βcatenin,wnt,Col Ⅱa,Col X expression.Results:1.The expression of miR-503-5p was low in rat epiphyseal chondrocytes and bone marrow mesenchymal stem cells stimulated by IL-1β.2.After transfection of miR-503-5p in rat epiphyseal chondrocytes,the expression of target protein DVL3 decreased,the expression of pathway-related index genes and proteins increased,and osteogenesis-related genes and proteins decreased;after knocking down DVL3,the expression of target protein DVL3 was further decreased,the expression of pathway-related index genes and proteins was increased,and osteogenesis-related genes and proteins was increased.After blocking Wnt/β-catenin signaling pathway by ICG-001,the expression of target protein DVL3 increased,the expression of pathway-related index genes and proteins decreased,and osteogenesis-related genes and proteins increased.Indicate that miR-503-5p reduces DVL3 expression by activating Wnt/β-catenin signaling pathway,thereby reducing endochondral ossification;3.The results above were also obtained by transfecting miR-503-5p,knocking down DVL3 and blocking Wnt/β-catenin signaling pathway in rat bone marrow mesenchymal stem cells;4.Those results showed that miR-503-5p reduced the expression of DVL3 by activating the Wnt/β-catenin signaling pathway to inhibit the osteogenic response of stem cells.5(1)In animal experiments,gross photography and DR imaging showed that the length of the operated limb in the IL-1β group was significantly shortened and thickened from 14 days after epiphyseal plate injury.Overexpression of miR-503-5p could reverse the shortening deformity,but the proximal tibia was still thickened.(2)At 7 days after epiphyseal plate injury,micro-CT results showed that bone bridge and bone hyperplasia began to appear at the edge of epiphyseal plate injury.At 28 days after injury,micro-CT results showed that bone bridge was formed in NS group without obvious deformity,bone bridge and deformity were formed in IL-1β group,physiological structure of bone bridge fibrous epiphyseal plate in agomir group was basically normal,bone bridge and cavity were formed in agomir-NC group.At the same time,threedimensional imaging analysis showed that BV/TV in IL-1βgroup was significantly increased than MI group at 28 days after injury(p<0.05).It is indicated that IL-1β can promote the ossification of injured epiphyseal plate.Although overexpression of miR-5035p cannot reduce the number of bone trabeculae,it can reduce the thickness and density of bone trabeculae,which can reduce the epiphyseal plate ossification caused by IL-1β to a certain extent.(3)qRT-PCR and Western blot results showed that local application of agomiR-5035p at 7 days after injury decreased the expression of IL-1β(p<0.05),increased the expression of miR-503-5p in epiphyseal cartilage(p<0.01),and the expression of DVL3 was increased(p<0.05),continued to 56 days after injury;the expression of Wnt/βcatenin related genes decreased in IL-1β group at 7 days after injury,and increased in miR503-5p overexpression group until 56 days after injury.From 7 days after injury,the bonerelated indexes of IL-1β group were significantly increased to 56 days after injury,which were decreased in miR-503-5p overexpression group,and the performance was the most obvious at 28 days after injury.(4)The results of HE staining and safranine fast green staining:inflammatory reaction period(1d)epiphyseal plate injury local inflammatory cells,necrotic cells gathered,each group had no significant difference;at the fiber formation stage(7d),the proliferation area and hypertrophic area of epiphyseal chondrocytes in the injury site of IL-1β group were disordered.The physiological structure of epiphyseal chondrocytes in the injury site of miR-503-5p group was normal and no bone trabeculae appeared.At the stage of bone bridge formation(14d),the infiltration of inflammatory cells in each group was less,the absorption of necrotic cells increased,and trabecular bone began to appear.The number of trabecular bone in IL-1β group was more and dense,and the number of trabecular bone in miR-503-5p group was less and evenly distributed.A stable bone bridge was formed during the shaping period(28d).56 days after operation,the bone bridge was stable.IL-1β destroyed the structure of local epiphyseal chondrocytes and promoted osteogenesis.MiR-503-5p could protect the structure of local epiphyseal chondrocytes and the distribution of trabecular bone.(5)Immunohistochemistry and immunofluorescence results showed that local application of IL-1β at each time point after epiphyseal plate injury decreased the expression of Col2a,increased the expression of Col X,increased the expression of BMP2,inhibited the proliferation of value-added areas,promoted cell hypertrophy in hypertrophic areas,disrupted the physiological structure of epiphyseal plate chondrocytes,affected physiological functions,increased the expression of DVL3,and decreased the expression of wnt.Immunofluorescence results suggested that IL-1β caused a decrease in the fluorescence expression of β-catenin;overexpression of miR-503-5p increased Col2a expression,decreased Col X expression,decreased BMP2 expression,inhibited IL-1βinduced epiphyseal chondrocytes disorder,activated wnt/β-catenin signaling pathway,inhibited osteogenic response,and reduced DVL3 expression.Conclusion:miR-503-5p reduced the expression of DVL3 by activating the Wnt/β-catenin signaling pathway,reduced the endochondral ossification reaction,and inhibited the osteogenic differentiation of bone marrow mesenchymal stem cells through the same signaling pathway and target protein.The central drilling injury of epiphyseal plate can increase the expression of IL-1β and decrease the expression of miR-503-5p in the injured area.The increase of IL-1β expression can cause ossification in the injured area and form bone bridge,resulting in limb shortening and angular thickening.Overexpression of miR503-5p can reverse the formation and deformity of bone bridge to a certain extent. |
PDF Full Text Request