Relationship Between DKK-1 Gene Promoter Methylation And Pathological Osteogenesis In Ankylosing Spondylitis

BackgroundAnkylosing spondylitis(AS)is a chronic systemic inflammatory disease of unknown etiology that predisposes to young men,mainly involving the spinal,sacroiliac joint,surrounding entheseal sites,as well as peripheral joints and extra-articular connective tissue including skin,gut,eyes,heart and lungs.The pathology of AS is characterized by repeated enthesitis.And the proliferation of inflammatory cells and their secretion of inflammatory cytokines leads to bone erosion,followed by neovascularization,aggregation of fibroblasts,and secretion of a large amount of cellular matrix leading to tissue fibrosis.Finally,under the action of osteoblasts and osteocytes,matrix mineralization leads to rigidity and permanent disability.Therefore,the key to the treatment of AS is to prevent or delay the pathological osteogenesis.At present,the mechanism of pathological ossification is still unclear.Numerous studies have shown that abnormal regulation of Wnt signaling pathway is a key factor in the pathogenesis of AS.Dickkopf-1(DKK-1)is a natural antagonist of Wnt signaling pathway.At present,a large number of studies have shown that DKK-1 down-regulation plays an important role in the pathological bone formation of AS,but the mechanism of DKK-1 down-regulation is not yet clear.DNA methylation is the most common branch of epigenetics,which binds methyl to cytosine through the catalysis of DNA methyltransferase(DNMT),resulting in gene silencing.DNA methylation is a switch that regulates gene expression and is involved in the occurrence and development of many diseases.But now there is no study on the relationship between DKK-1 methylation and AS.ObjectiveTo investigate the mechanism of down-regulation of DKK-1 that can lead to pathological bone formation in AS from the perspective of genetic epigenetics.We detected the methylation status of DKK-1 gene promoter and the expression of DKK-1 and DNMTs in the ossified joint capsule of AS patients and normal hip joint capsule to find the relationship between DKK-1 gene promoter methylation and pathological osteogenesis in AS.MethodsThe synoviums of ossified hip capsules were collected from AS patients underwent hip arthroplasty,hip arthroplasty or hip lesion excision,and the synoviums of normal hip joint capsules of patients with femoral neck fracture(FNF)caused by trauma who underwent hip replacement surgery were collected as control in the Department of Joint surgery or Sports Medicine in our hospital from July 2017 to January 2020.The expression of DKK-1 protein was detected by Western Blot.The methylation status of DKK-1 gene promoter was detected by methylation-specific PCR(MSP),and the expressions of DNMTs mRNA were detected by real-time quantitative PCR(qPCR).ResultsThe expression of DKK-1 protein in the AS group was significantly lower than that in FNF group,and there was a significant difference between the two groups(P<0.05).DKK-1 gene in all patients in the FNF group was unmethylated,with a methylation rate of 0,while the AS group showed partial methylation and complete methylation,with a methylation rate of 1.There was significant difference between the two groups(P<0.05).Compared with the FNF group,the expression of DNMT1 in the AS group was decreased(P=0.71),and the expression of DNMT3A and DNMT3B was increased(P=0.38,P=0.46).But there was no significant difference in DNMTs between the two groups(P>0.05).ConclusionThe hypermethylation of DKK-1 gene promoter inhibits the expression of DKK-1 and promotes the pathological osteogenesis in AS,which provides clues for innovative diagnosis,biomarker for evaluating disease progression or drug efficacy and new targeted therapy of AS.