| In recent years,the innovation of artificial prosthesis design and the improvement of surgical techniques have made the postoperative effect more ideal.However,the etiology of non-mechanical,aseptic loosening of the hip prosthesis has not been determined,although the problems of material wear and infection around the prosthesis have been well resolved.Some scholars believe that the imbalance of osteogenesis and osteolysis around the prosthesis is the main cause of aseptic loosening of the prosthesis,but its pathogenesis has not been fully elucidated.In recent years,circ RNAs have been widely reported as a new type of non-coding RNA and are believed to be closely related to many diseases.More and more research evidence suggests that circ RNAs,as competitive endogenous RNAs(ce RNAs),can bind to micrornas(mi RNAs)and regulate their downstream functions.At present,it has been found that circ RNAs play a crucial role in the pathogenesis,diagnosis and targeted therapy of some degenerative diseases(such as osteoarthritis),metabolic diseases(such as osteoporosis)and bone tumors(such as osteosarcoma).Previous studies of our team have shown that circ RNA may be related with some biological functions and signal pathways of prosthetic osteolysis,and we firmly believe that further study of circ RNA-mi RNA-m RNA signaling pathway will reveal new pathogenesis and treatment strategies of prosthesis osteolysis.This study consists of the following three parts:Part I: Study on the expression of circ＿0000690 in osteoblasts in the microenvironment of periprosthetic osteolysis(PPO)Objective: To analyze the tissue samples of patients with PPO of artificial hip prosthesis by high-throughput sequencing.q RT-PCR was used to verify the expression of circ＿0000690 in PPO patients with artificial hip prosthesis and osteoblast cell lines co-cultured with titanium particles in vitro.Bioinformatics analysis was performed to predict mi RNA and m RNA downstream of circ＿0000690.Methods: In this study,bone tissue around the prosthesis of 12 patients with PPO after total hip arthroplasty was collected as the experimental group,and bone tissue at the fracture end of 12 patients undergoing primary hip arthroplasty due to femoral neck fracture was collected as the control group.High-throughput sequencing analysis was performed on collected clinical specimens to screen out circ RNAs that expressed significantly differently in PPO tissues of the prosthesis after total hip replacement.Further,q RT-PCR was used to verify the expression of circ RNA in patients with PPO of artificial hip prosthesis and osteoblast cell lines co-cultured with titanium particles in vitro.Meanwhile,bioinformatics analysis was used to predict the downstream mi RNA and m RNA,and the ce RNA regulatory network diagram was mapped.Results: A total of 257 circ RNAs were identified by high-throughput sequencing with significant differences in the expression of circ RNAs in the tissues of patients with PPO after total hip replacement,and the expression of circ＿0000690 was significantly down-regulated.The downstream mi RNA and m RNA were predicted by Target Scan and Circular RNA Interactome,and the ce RNA regulatory network was mapped.q RT-PCR results confirmed that circ＿0000690 expression was significantly down-regulated in PPO patients with artificial hip prosthesis and in osteoblast cell lines co-cultured with titanium particles in vitro.Conclusion: The expression of circ＿0000690 was inhibited in the PPO bone tissue.Part II: Study on the role of circ＿0000690 in regulating osteoblast apoptosis Objective: To investigate the effect of circ＿0000690 on the biological behavior of human osteoblast cell line in vitro.Methods: Circ＿0000690 overexpressed plasmid and si RNA fragment were synthesized and transfected into human osteoblasts to verify the effect of circ＿0000690 on the biological behavior of human osteoblast cell lines in vitro.Results: Compared with the negative control group,the proliferation ability of human osteoblasts in circ＿0000690 overexpression group was significantly increased,the proportion of osteoblasts apoptosis was significantly decreased,the expression of apoptosis-related proteins was significantly down-regulated,and the expression of anti-apoptotic proteins and cyclin was significantly up-regulated.In circ＿0000690knockout group,the proliferation ability of human osteoblasts was significantly reduced,the apoptosis ratio of osteoblasts was significantly increased,the expression of apoptosis-related proteins was significantly up-regulated,and the expression of anti-apoptotic proteins and cyclin was significantly down-regulated.Conclusion: Circ＿0000690 can participate in the positive regulation of osteoblast proliferation and inhibit osteoblast apoptosis.Part III: Study on the molecular mechanism of circ＿0000690/Mir-361-3p/RHBDF2 in regulating osteoblast apoptosisObjective: To explore the molecular mechanism of circ＿0000690 affecting the biological function of osteoblast cell lines cultured in vitro.Methods: The subcellular localization of circ＿0000690 was verified by FISH test.Bioinformatics databases such as Circular RNA Interactome and Target Scan were used to predict the interaction between circ＿0000690 and mir-361-3p and target gene RHBDF2,and the targeted binding of circ＿0000690 and mir-361-3p was verified by double luciferase reporter assay.Immunohistochemistry was used to confirm the expression of RHBDF2 in patients with PPO after THA.The expression of mir-361-3p and RHBDF2 in patients with PPO was detected by realtime-PCR and the correlation between mir-361-3p and RHBDF2 and circ＿0000690 was analyzed.Realtime-PCR and Western Blot were used to detect the effect of circ＿0000690expression on the expression of mir-361-3p and RHBDF2.The effect of mir-361-3p expression changes on RHBDF2 expression was detected.Cell Rescue experiment verified that circ＿0000690 acts as a molecular sponge to adsorb mir-361-3p,and participates in regulating the biological behavior of osteoblast apoptosis through circ＿0000690/ mir-361-3p /RHBDF2 axis.Results: FISH test indicated that circ＿0000690 was mainly located in cytoplasm.RNase R test confirmed the existence of circ＿0000690 ring structure.Mir-361-3p is the downstream target gene of circ＿0000690: Bioinformatics predicted that circ＿0000690 could target mir-361-3p,mir-361-3p was highly expressed in patients with PPO after THA,and was negatively correlated with circ＿0000690.Dual luciferase reporter gene detection further confirmed the targeted binding of the two.Overexpression of circ＿0000690 can down-regulate mir-361-3p expression.RHBDF2 is a downstream target gene regulated by mir-361-3p.Immunohistochemical results confirmed that RHBDF2 was significantly underexpressed in patients with PPO after THA.Bioinformatics predicted that mir-361-3p and RHBDF2 could be targeted to bind and had a common binding site with circ＿0000690,and circ＿0000690 and RHBDF2 could compete to bind to mir-361-3p.RHBDF2 gene was significantly lower expressed in patients with PPO after THA,and was significantly negatively correlated with the expression of mir-361-3p.Double luciferase reporter gene further confirmed the targeting relationship between the two,and overexpression of mir-361-3p could inhibit the expression of RHBDF2.Circ＿0000690 regulates the biological behavior of PPO after THA by influencing the expression of RHBDF2 through the adsorption of mir-361-3p:The expression level of circ＿0000690 was positively correlated with RHBDF2 in patients with PPO after THA,and overexpression of circ＿0000690 could promote the expression of RHBDF2.Co-transfection of mir-361-3p mimics inhibited the up-regulation of RHBDF2 expression by circ＿0000690 overexpression.Co-transfection of mir-361-3p mimics inhibited the effects of circ＿0000690overexpression on proliferation,apoptosis and cycle of human osteoblasts.Co-transfection of mir-361-3p mimics inhibited the effects of circ＿0000690overexpression on apoptosis and expression levels of cycle-related proteins Cleaved caspase-3,Bcl-2,and cyclin D1.Conclusions: In osteoblasts,circ＿0000690 acts as a molecular sponge,competitively inhibiting mir-361-3p,and then positively regulating the target gene RHBDF2,participating in inhibiting osteoblast apoptosis through circ＿0000690/mir-361-3p/RHBDF2 pathway.General conclusion: Circ RNA circ＿0000690 negatively regulates osteoblast apoptosis through circ＿0000690/mir-361-3p/RHBDF2 pathway and participates in the biological effects of PPO. |
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