| Total hip arthroplasty is an effective treatment for advanced hip arthritis,which can significantly reduce pain and improve hip function.As a routine procedure,total hip arthroplasty is widely performed worldwide.Postoperative aseptic loosening of prosthesis is one of the most serious complications in hip arthroplasty.For aseptic loosening of prosthesis after hip replacement,revision of prosthesis is an effective method at present.However,revision surgery is not only relatively risky,but also relatively complex and expensive,and most importantly,the postoperative results of revision surgery are usually not particularly ideal.Therefore,it is very important to reduce the incidence of aseptic loosening and revision rate of hip prosthesis.At present,it is believed that the main pathogenic factor for aseptic loosening of prosthesis is the wear of prosthesis particles,which is the theory of particle disease.In addition,there are many factors involved,such as prosthesis freting,stress blocking,changing of joint fluid pressure,endotoxin and so on.Although causes vary,aseptic loosening is ultimately caused by periprosthetic osteolysis.The initial stability of the biological prosthesis mainly depends on the pressing of the prosthesis and bone,while the long-term stability mainly depends on the surface bone insertion of the prosthesis.When the prosthesis is implanted,a variety of cytokines are released to induce osteoblasts to differentiate and mature,and osteoblasts form new bone tissue on the surface of the prosthesis,thus making the prosthesis firmly fixed.When the activity of osteoblasts around the prosthesis is inhibited,new bone cannot be formed on the surface of the prosthesis,and the activity of osteoclasts is enhanced,which will lead to osteolysis around the prosthesis and aseptic loosening of the prosthesis.The mechanism of osteoblasts activity and maturation is complex and varied.If the specific regulation mechanism of osteoblasts activity and maturation during aseptic loosening of prosthesis can be revealed,it will provide a theoretical basis for the prevention and treatment of aseptic loosening.Current studies have shown that circRNA is closely related to the occurrence and development of a variety of diseases,including osteoarthritis and osteoporosis.In addition,studies have shown that circRNA can regulate the activity of osteoblasts,so we speculated that circRNA may be closely related to aseptic hip prosthesis loosening.We then performed high-throughput sequencing on aseptic loosening tissues of hip prosthesis,screened for differentially expressed circRNAs,and studied the related functions and mechanisms.This research consists of the following three parts.Part ?The differentially expressed circRNAs in the aseptic loosened process and the ceRNA regulatory axis Objective: This study used prosthesis surrounding tissues from 6 patients with hip prosthesis aseptic loosening and hip joint synovial tissue from 6 patients undergo first time artificial hip replacement for high-throughput sequencing analysis.We screened differentially expressed circRNAs,and determined target circRNA by qRT-PCR.Based on the target circRNA,ceRNA regulation axis was biulded to find new biomarkers for the diagnosis and treatment of hip prosthesis aseptic loosening.Methods: Clinical tissue samples were collected,total RNA was extracted,and high-throughput sequencing library was constructed.High-throughput sequencing was used to analyze the sequencing data,and circRNAs with significant differential expression were screened out.The GO and KEGG pathway enrichment analysis was performed on host genes and target genes of differentially expressed circRNAs by David database.PCR verified the 10 circRNAs with the most obvious differential expression.Based on Target Scan and miRanda database,the targeted miRNA and mRNA of the target circRNA were predicted,and the ceRNA regulatory axis was constructed.Results: High-throughput sequencing showed 257 circRNAs differentially expressed in aseptic loosening tissues,including 171 circRNAs up-regulated and 86 circRNAs down-regulated.There were 1789 mRNA up-regulated and 2142 mRNA down-regulated in aseptic loosening tissues.KEGG analysis results showed that the host genes of differentially expressed circRNAs ware mainly enriched in epithelial cells of bacteria invasion,regulation and control of actin cytoskeleton,vasopressin adjustment of water absorption and protein,polysaccharide,PI3K-Akt signaling pathways in cancer,Rap1 signaling pathways,prostate cancer,autophagy,synthesis and secretion of cortisol,and adrenergic signaling pathways in myocardial cells.KEGG analysis results showed that target genes of circRNAs ware mainly enriched in the transcription regulation pathways of cancer,kaposi's sarcoma associated herpesvirus infections,chemokine signaling pathways,toll-like receptors signaling pathways,autoimmune thyroid disease,epithelial cells of bacteria invasion,primary immunodeficiency,thyroid cancer,adhesive connection,and thyroid hormone synthesis.Based on the qRT-PCR validation of ten most differentially expressed circRNAs,hsa-circ-0005232 was identified as target circRNA for our study,and the hsa-circ-0005232/hsa-miR-21-3p/ULK1 regulatory axis was constructed.Conclusions: We performed high-throughput sequencing on the tissues surrounding aseptic loosening prosthesis and normal tissues during first time total hip replacement,and found 257 differentially expressed circRNAs,among which hsa-circ-0005232 was significantly down-regulated.We constructed the hsa-circ-0005232 /hsa-miR-21-3p/ULK1 regulatory axis using bioinformatics methods.Our findings will be helpful for the study of the pathogenesis of aseptic loosening and the search of new therapeutic targets.Part ? The regulation effect of hsa-circ-0005232 on the activity and maturation Objective: In the first part,we found that hsa-circ-0005232 was significantly down-regulated in aseptic prosthesis loosening tissues,and studies have shown that hsa-circ-0005232 is associated with a variety of bone diseases.We examined the effect of hsa-circ-0005232 on the activity and maturation of osteoblasts in a cell model.Methods: First,hsa-circ-0005232 high expression and knockdown lentivirus were constructed and transfected into human osteoblasts.The expression of hsa-circ-0005232 and ULK1 in osteoblasts was detected by qRT-PCR.The autophagosome formation of osteoblasts was observed by electron microscopy,and the protein expressions of LC3 ?,LC3 I and ULK1 in osteoblasts were detected by Western blot.The expressions of alkaline phosphatase,osteocalcin,type I collagen and calcium nodule formation were also detected.Results: ULK1 expression was elevated when hsa-circ-0005232 was overexpressed,and decreased when hsa-circ-0005232 was low.Electron microscopy results showed that the number of autophagosomes was significantly increased in the hsa-circ-0005232 overexpression group,while the number of autophagosomes was significantly decreased in the knockdown group.Therefore,the experimental results showed that hsa-circ-0005232 could significantly increase the level of autophagy in osteoblasts.Compared with the control group,LC3 ?/ LC3 I was significantly increased in the overexpression group of hsa-circ-0005232,and the ULK1 protein band was also significantly enhanced.However,LC3 ?/ LC3 I and ULK1 protein expression were significantly reduced in the hsa-circ-0005232 knockdown group.Alkaline phosphatase was significantly increased in the high expression group of hsa-circ-0005232,while alkaline phosphatase was significantly decreased in the knockdown group of hsa-circ-0005232 compared with the control group.The secretion of osteocalcin in the hsa-circ-0005232 high-expression group was significantly higher than that in the control group,while the secretion of osteocalcin in the hsa-circ-0005232 knockdown group was significantly lower.The expression of ? type collagen in the overexpression group of hsa-circ-0005232 was higher than the control group obviously,and the ? type of collagen content in the hsa-circ-0005232 knockdown group is significantly lower than the control group.The formation of calcium nodules in hsa-circ-0005232 overexpression group was significantly higher than that in the control group,while the formation of calcium nodules in hsa-circ-0005232 overknockdown group was significantly lower than that in the control group.These results indicated that hsa-circ-0005232 could significantly promote the activity and maturation of osteoblasts.Conclusions: Hsa-circ-0005232 can affect autophagy by regulating ULK1 expression,thereby regulating the activity and maturation of osteoblasts.These results suggest that hsa-circ-0005232 may participate in the development of aseptic loosening by regulating the surface bone insertion of prosthesis.Part ? hsa-circ-0005232 regulates the expression of ULK1 through competitive inhibition of hsa-miR-21-3p Objective: We have determined that hsa-circ-0005232 is significantly down-regulated in aseptic hip prosthesis loosening,and constructed the hsa-circ-0005232/ hsa-miR-21-3p/ULK1 regulatory axis,and further studies have shown that hsa-circ-0005232 can regulate autophagy to affect activity and maturation of osteoblasts through ULK1.However,the regulatory mechanism of hsa-circ-0005232 on ULK1 remains to be further clarified.Methods: We constructed a recombinant double luciferase reporter assay to test the binding ability of hsa-circ-0005232 to hsa-miR-21-3p and ULK1 to hsa-miR-21-3p.We constructed hsa-circ-0005232 and hsa-miR-21-3p overexpressing lentiviruses,which were transfected into osteoblasts,and the expression of target gene ULK1 was verified by qRT-PCR.Results: The luciferase activity of the co-transfection group with hsa-circ-0005232-WT and hsa-miR-21-3p mimics was significantly lower than that of the negative control group,while the luciferase activity of the co-transfection group with hsa-circ-0005232-mut and hsa-miR-21-3p mimics was not significantly different from that of the negative control group.The luciferase activity of the co-transfection group with ULKi-WT and hsa-miR-21-3p mimics was significantly lower than that of the negative control group.There was no significant difference in luciferase activity between the ULK1-mut and hsa-miR-21-3p mimics co-transfection group and the negative control group.These results indicated that both hsa-circ-0005232 and ULK1 could directly bind to hsa-miR-21-3p.ULK1 mRNA expression was significantly increased when hsa-circ-0005232 was overexpressed,while ULK1 expression was significantly decreased when hsa-miR-21-3p was overexpressed.The inhibitory effect of hsa-miR-21-3p on ULK1 expression could be reversed by overexpression of hsa-circ-0005232.These results indicated that hsa-circ-0005232 indirectly regulated the expression of target gene ULK1 through competitive inhibition of hsa-miR-21-3p.Conclusions: Hsa-circ-0005232 could indirectly regulate the expression of ULK1 mRNA through competitive inhibition of hsa-miR-21-3p,and play the role of ceRNA. |
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