| Objective: Hip osteoarthropathy,aseptic necrosis of femoral head,hip fracture and other hip diseases in the elderly can lead to hip pain and limited movement.At present,hip replacement is widely used in the end-stage treatment of hip diseases,which can effectively reduce hip pain and restore hip function as soon as possible,so the number of artificial joint replacement is increasing year by year.However,periprosthetic osteolysis is the main reason why revision surgery is still required for joint prostheses.The pathological basis of prosthesis loosening is periprosthetic osteolysis,which is caused by the interaction between the wear particles produced at the prosthesis-bone interface and the periprosthetic tissues.Under the induction of wear particles,osteoblasts,osteoclasts and mononuclear macrophages around the prosthesis produce a large number of cytokines and chemokines,which lead to a series of cellular reactions,such as chronic inflammation,apoptosis and so on,and destroy the balance of bone metabolism,eventually leading to osteolysis and aseptic loosening.With the further research of circRNAs in orthopedic related fields,researchers have found that circRNAs have important reference significance in the pathogenic mechanism,diagnosis and gene targeted therapy of orthopedic related diseases.However,there are few reports on the role of circRNAs in the loosening of artificial joint prostheses.Based on the existing work,we analyzed the differential expression of circRNAs and target gene m RNA in the tissues of 10 sterile loosening patients and 10 non-sterile loosening hip joints by high-throughput sequencing.Differentially expressed circRNAs were screened.Among them,hsa＿CIRC＿0007482 was significantly up-regulated.The aim of this study was to construct a ce RNA grid,explore the expression of CIRC RNA0007482 in osteoblasts induced by Ti particles,and reveal new potential mechanisms and therapeutic strategies of osteolysis around artificial joints.The downstream miRNA and m RNA of circ-0007482 were predicted by bioinformatics analysis,and colluded with ce RNA grid.RT-PCR was used to verify the expression of circ-0007482 in osteoblasts co-cultured with Ti particles in vitro and induced osteoblasts apoptosis model.The biological function of circ-0007482 was further verified.According to the ce RNA grid constructed in the first part of the experiment,the mechanism of circ-0007482 participating in osteolysis by adsorbing miRNA-511-5p sponge and regulating MAP4K4 was verified.Methods: Target Scan database and miRanda database were used to predict the downstream miRNA and m RNA.The m RNA which can be up-regulated by binding with the miRNA is detected,go enrichment and KEGG analysis are carried out according to the potential interaction in the grid of the circRNA/miRNA/m RNA,and the molecular biological function of the circ-0007482 and the signal pathway involved in regulation are screened.RT-PCR verification of circ-0007482 in Ti particles and h FOB 1.19 cells were co-cultured to establish osteoblast apoptosis model induced by Ti particles.The apoptosis rate of osteoblasts induced by Ti particles was detected by TUNEL and flow cytometry after circ-0007482 silencing,the expression level of apoptosis-related genes was detected by RT-PCR,and the expression level of apoptosis-related proteins was detected by western blot.In order to verify the molecular mechanism of circ-0007482/miRNA-511-5p/MAP4K4 involved in osteoblast apoptosis,the subcellular localization of circ-0007482 was verified by FISH.Dual-luciferase reporter validated the targeted binding of circ-0007482 to miRNA-511-5p and miRNA-511-5p to MAP4K4.RIP experiment verified that circ-0007482 and miRNA-511-5p can be directly combined.TUNEL and flow cytometry were used to detect the effects of silencing and overexpression of circ-0007482 on osteoblast apoptosis induced by Ti particles,and RT-PCR was used to detect the expressions of miRNA-511-5p,MAP4K4 and apoptosis-related genes.The expression levels of apoptosis-related proteins,p38 MAPK and p-p38 MAPK were detected by western blot.To verify the effect of miRNA-511-5p on MAP4K4 and the apoptosis rate of osteoblasts induced by Ti particles.The expression levels of apoptosis-related genes and proteins were detected by RT-PCR and western blot.Finally,through the recovery experiment,circ-0007482 and miRNA-511-5p were co-transfected to verify the mechanism of ce RNA,and to detect the effect of Ti particles on osteoblast apoptosis in the model of osteoblast apoptosis.Results: In the first part,according to the results of bioinformatics analysis,the circ-0007482/miRNA-511-5p/MAP4K4 grid was established.RT-PCR showed that the expression of circ-0007482 was significantly up-regulated in the osteoblast apoptosis model induced by Ti particles in vitro.After Silencing circ-0007482,the apoptosis rate of osteoblasts detected by TUNEL and flow cytometry was significantly lower than that of Ti particle induction group and silencing blank group,while the expression of apoptosis-related genes Bax and Caspase-3 was down-regulated,and the relative expression of anti-apoptotic gene Bcl-2 was up-regulated.The expression level of apoptosis-related proteins was consistent with the above trend.In the second part,Fish experiment confirmed that circ-0007482 was located in the cytoplasm,and dual-luciferase reporter gene confirmed that circ-0007482 could target and bind to miRNA-511-5p,and miRNA-511-5p could target and bind to MAP4K4.RIP experiment confirmed that circ-0007482 could directly bind to miRNA-511-5p.In the apoptosis model induced by Ti particles,the expression of circ-0007482 and MAP4K4 was up-regulated,and the expression of miRNA-511-5p was down-regulated,which was consistent with the basic trend of ce RNA.Overexpression of circ-0007482 increased the apoptosis rate of osteoblasts compared with the Ti particle induced group and the blank overexpression group.The expression of Bax,Bcl-2 and caspase-3 was detected by RT-PCR.Protein levels of Bax,Bcl-2,Caspase-3 and mitogen-activated protein kinases(MAPKs)were detected by western blot.The phosphorylation level of p38 MAPK,Bax,Caspase-3 and Bcl-2 protein were consistent with the trend of PCR,and the phosphorylation level and activation of p38 MAPK were increased.When circ-0007482 was silenced,the apoptosis rate of osteoblasts decreased,the levels of apoptosis-related genes detected by RT-PCR and the levels of apoptosis-related proteins detected by western blot were parallel to the trend of apoptosis,and the phosphorylation of p38 MAPK decreased compared with the silencing blank group.To verify the molecular regulation mechanism of miRNA-511-5p,miRNA-511-5p inhibitor and mimics were transfected,respectively.Transfection of miRNA-511-5p mimics could reduce osteoblast apoptosis induced by Ti particles,and TUNEL and flow cytometry confirmed that the apoptosis rate of osteoblasts transfected with miRNA-511-5p mimics was significantly lower than that of the transfected mimics blank group.The expression of Bax and Caspase-3 genes in the transfection mimic group was significantly down-regulated compared with the blank group,and the expression of Bcl-2 gene was significantly up-regulated.The expression level of apoptosis-related proteins detected by western blot was consistent with that detected by RT-PCR,and the phosphorylation level of p38 MAPK was lower than that in the blank group.Transfection with the miRNA-511-5p inhibitor further increased osteoblast apoptosis.The apoptosis rate of osteoblasts detected by TUNEL and flow cytometry was significantly increased compared with the inhibitor blank group,and the results of RT-PCR showed that the expression levels of Bax and Caspase-3 genes in the inhibitor group were significantly up-regulated compared with the blank group,and the expression level of Bcl-2 was significantly down-regulated.The results of western blot showed that the expression level of apoptotic protein in the inhibitor group was consistent with the results of RT-PCR.The phosphorylation level of p38 MAPK in the inhibitor group was higher than that in the blank group.To further confirm the regulatory mechanism of circ-0007482/miRNA-511-5p/MAP4K4 axis in osteoblast apoptosis induced by Ti particles,a reversion experiment was performed.The si-circ-0007482 and miRNA-511-5p inhibitor were separately and co-transfected into the Ti-induced osteoblast apoptosis model.TUNEL and flow cytometry were used to detect the apoptosis rate of osteoblasts,PCR was used to detect the expression of MAP4K4 and apoptosis-related genes,and western blot was used to determine the level of apoptosis-related proteins and the expression of p38 MAPK and p-p38 MAPK.The results showed that the expression of MAP4K4 protein was decreased by transfection of si-circ-0007482 alone,and increased by transfection of miRNA-511-5p inhibitor alone.The inhibitory trend of MAP4K4 expression was significantly reversed.The expression of MAP4K4 was significantly up-regulated after transfection of circ-0007482 alone;The expression of MAP4K4 was down-regulated after transfection of miRNA-511-5p inhibitor alone,and the expression of MAP4K4 was down-regulated after transfection of miRNA-511-5p mimics with overexpression of circ-0007482,which was statistically significant.Conclusion: In osteoblasts,circ-0007482 plays the role of molecular sponge,competitively inhibits miRNA-511-5p,positively regulates and controls target gene MAP4K4,and promotes osteoblast apoptosis through circ-0007482/miRNA-511-5p/MAP4K4 pathway.Involved in periprosthetic osteolytic effects. |
