| Objective:Periprosthetic osteolysis(PPOL)is the leading cause of total joint arthroplasty(TJA)failure.The revision rate is expected to increase by 137%by 2030.PPOL accounted for more than 51%of all revision cases causing a huge public health burden worldwide.Further solutions are urgently needed.Bone remodeling is a dynamic process of bone destruction and bone reconstruction,that is,bone destruction and absorption induced by osteoclasts and new bone formation participated by osteoblasts,and bone formation and bone absorption are dynamically regulated to maintain bone homeostasis.PPOL is closely related to bone homeostasis.However,the release of wear particles from the sliding interface of the prosthesis triggers the increase in osteoclast activity,leading to the dysregulation of bone homeostasis and eventually resulting in the PPOL.PPOL is the final result of chronic progressive inflammatory reaction at the bone-prosthesis interface.Non-coding RNA(ncRNA)accounts for about 98%of the total RNA,and participates in the editing and modification of RNA,protein translation and other biological functions.At present,some nc RNAs have been found to regulate the occurrence and development of the bone and joint diseases and bone tumors.Our previous studies have shown that hsa＿circ＿0000994(circ＿994)is significantly inhibited in the periprosthetic osteolytic boundary membrane tissue.To further study the pathogenesis of periprosthetic osteolysis,we employed bioinformatics to predict the downstream regulatory genes of circ＿994 and the molecular pathways involved in their regulation.By analyzing the relationship between circ＿994 and the increase in osteoclastic activity,we gained new insight into the methods for treating periprosthetic osteolysis.Methods:1.The microarray data of miRNA expression related to osteoclast activation GSE63773and the m RNA transcriptome data GSE171542 were screened by bioinformatics tools.Differential expression analysis of miRNA or m RNA was performed using"limma"packets.The circ RNA-miRNA-m RNA binding sites were predicted through the online platforms circ Bank,Star Base,and targetscan databases.2.Boundary membrane tissue samples of the revision of the THA from patients(PPOL group)and the synovial tissue samples of the hip joint from patients with femoral neck fracture during the initial THA(Normal group)were collected.Circ＿994 and miR-409-3p gene expression in different groups was detected by q RT-PCR.The expression of target protein PLXDC2,the markers of osteoclast activation(MMP8,MMP9,RANKL,TRAP),and inflammatory factors(IL-1β,IL-6,and TNF-α)in macrophages was examined by q RT-PCR,Western blot,and immunohistochemistry(IHC).H&E was used to determine the infiltration of inflammatory cells and multinucleated osteoclasts in tissues.Alizarin red staining was used to detect the osteogenesis.Immunofluorescence staining was used to detect the infiltration of inflammatory macrophages in tissues.3.In in vitro experiments,THP-1 cells were induced into macrophages by PMA,and osteoclast-like cells were induced by the macrophage colony-stimulating factor(M-CSF)combined with RANKL.Si-circ＿994 was used for knocking down circ＿994.For constructing the overexpressed circ＿994,the whole circular RNA and artificial reverse repeat sequence were cloned into pc DNA3.1 vector and transfected into the THP-1 cells stimulated with RANKL/M-CSF.miR-409-3p mimic,miR-409-3p inhibitor and negative control miRNA mimic(mimic NC)were transfected into THP-1 cells to construct the miR-409-3p overexpression,knockdown,and control cell lines,respectively.The interactions between circ＿994 and miR-409-3p,as well as miR-409-3p and PLXDC2,were verified by the luciferase reporter and fluorescence in situ hybridization(FISH)assays,respectively.Stimulate(Ti)macrophages with Ti particles or use targeted circ＿994(si-circ＿994)si RNA was transfected into macrophages.The expression of circ＿994/miR-409-3/PLXDC2 signal axis-related index genes and proteins,the expression of inflammatory factors secreted by macrophages,and osteoclast activation-related marker proteins were detected by q RT-PCR or Western blot.4.Human-derived circ＿994 was converted to murine mmu＿circ＿994.Mmu＿circ＿994overexpressing and mmu-miR-409-3p mimic overexpressing macrophage cell lines were constructed.Expression of mmu-miR-409-3p and PLXDC2 in the cell lines was confirmed using q RT-PCR.The constructed overexpressing cells were collected and injected into the joint cavity of mouse osteolysis models,with Ti inserted into the tibial osteolysis model.To evaluate the bone loss,micro-CT was used to assess bone volume(BV),bone volume fraction(BV/TV),bone surface/volume ratio(BS/BV),trabecular number(Tb.N),trabecular thickness(Tb.Th),and trabecular separation(Tb.Sp).The roles of mmu＿circ＿994 and mmu-miR-409-3p were verified in mouse osteolysis models.Results:1.Genes associated with osteoclast activation were screened by bioinformatics analysis using the microarray data GSE63773 and m RNA transcriptome data GSE171542.Using the online platform circ Bank,Star Base,and targetscan databases,circ＿994/miR-409-3p/PLXDC2 was predicted to be the critical signal axis for regulating the osteoclastic activation of macrophages.2.The human specimen experiment revealed significant differences between the PPOL and normal groups.Specifically,circ＿994 expression was lower in the boundary membrane tissue of patients in the PPOL group.Moreover,q RT-PCR showed that the relative m RNA expression of PLXDC2 m RNA was decreased,while that of MMP8,MMP9,and RANKL genes,which are related to macrophage inflammation and osteoclast activation,was increased,consistent with Western blot results.Immunohistochemical staining demonstrated higher expression levels of multiple cytokines in the PPOL group,including MMP8,MMP9,TNF-α,IL-1β,and IL-6.The lower expression of PLXDC2 in the PPOL group was associated with increased infiltration of inflammatory cells,multinucleated osteoclast,and macrophages(CD68~+)in tissues,as evidenced by IHC and H&E staining.In addition,the PPOL group had increased infiltration of inflammatory macrophages(CD68~+/i NOS~+),which may be related to reduced osteogenesis.3.In in vitro experiments,luciferase reporter and FISH assays confirmed the interactions between circ＿994 and miR-409-3p,as well as miR-409-3p and PLXDC2.Circ＿994 and miR-409-3p were co-localized in the cytoplasm.Compared with the control group,the relative expression of circ＿994 in cells of the Ti group decreased,further promoting the expression of miR-409-3p,as well as the macrophage inflammation-related genes,i.e.,IL-1β,IL-6,and TNF-α.At the same time,the expression of MMP8,MMP9,RANKL,and TRAP was increased,and the expression of PLXDC2 was decreased in cells of the Ti group;these changes were also observed in cells of the si-circ＿994 group.The overexpression of circ＿994 in macrophages exposed to Ti particles reversed the increase of miR-409-3p.The overexpression of circ＿994 had a specific reversal effect on the expression of inflammatory genes in macrophages and the differentiation of osteoblasts,which was mediated by promoting the expression of PLXDC2.Rescue experiments showed that miR-409-3p participated in the circ＿994-mediated osteoclast activation process,and the expression of PLXDC2 in macrophages transfected with miR-409-3p mimic was downregulated.The expression of the macrophage inflammation-related proteins IL-1β,IL-6,and TNF-αand the osteoclast activation-related proteins MMP8,MMP9,RANKL,and TRAP increased;these changes were consistent with those in cells of the Ti group.However,the inhibition of miR-409-3p increased the expression of PLXDC2 and reversed the increase in the expression of macrophage inflammation-related factors and osteoclast activation-related factors induced by Ti particles and si-circ＿994.4.Mouse animal experiments verified the decrease of mmu-miR-409-3p expression and the increase of PLXDC2 expression in mouse macrophages over-expressing mmu＿circ＿994;the overexpression of mmu＿circ＿994 attenuated periprosthetic osteolysis in mouse models induced by Ti particles.Transfection of macrophages overexpressing mmu＿circ＿994 with mmu-miR-409-3p mimic downregulated the expression of mmu＿circ＿994 and PLXDC2.In the mouse osteolysis model transfected with mmu＿circ＿994,BV,BV/TV,Tb.N,and Tb.Th were significantly increased,while BS/BV and Tb.Sp was significantly decreased.In the mouse osteolysis model transfected with mmu＿circ＿994 and mmu-miR-409-3p,BV and BV/TV were significantly increased,BS/BV was significantly decreased,Tb.N and Tb.Th were significantly increased,Tb.Sp was significantly decreased,BS/BV was significantly increased,Tb.N and Tb.Th were significantly decreased,Tb.Sp was significantly increased.Conclusion:Circ＿994/miR-409-3p/PLXDC2 forms a ce RNA network axis.Circ＿994 negatively regulates osteoclast activation induced by Ti particle through the miR-409-3p/PLXDC2pathway and participates in the pathological process of periprosthetic osteolysis. |
PDF Full Text Request