Differential Expression Of Kaiso Antibody In The Blood Of Patients With Ankylosing Spondylitis And Its Effect On The Process Of Ossification And The Underlying Mechanisms

BackgroundAnkylosing spondylitis(AS)is widely considered as a chronic,debilitating,inflammatory disease,characterized by enthesitis,progressive spinal ankylosis and sacroiliitis.The disease occurs mostly in young people and has a high incidence.Due to the lack of typical early symptoms and early biomarker for diagnosis,it usually gets a clear diagnosis after the first clinical symptom has occurred for 8 to 10 years.In patients with advanced AS,spinal ankylosis and hip bone fusion will cause the patients to lose the ability of living and working which is a great burden on families and society.The ossification of the spine and joints are the main pathological features of AS,the mechanism of its occurrence and development is still unclear.Therefore,searching early diagnostic markers for AS and exploring the biological mechanisms of ossification have been the focus of AS research.Autoantibodies are immune markers of many autoimmune diseases and can be used as early diagnoses,observations of disease activity,assessment of prognosis of treatment.Ankylosing spondylitis has long been considered a seronegative spondyloarthropathy due to the lack of rheumatoid factor,and sera autoantibodies are seldom considered as biomarkers.However,there are more and more evidences that B cells and corresponding humoral immune responses may participate in the pathogenesis of AS through antigen presentation,antibody production and the like.Therefore,this study attempts to screen the plasma of AS through the human proteome microarrays to find autoantibodies suitable for early diagnosis of AS and further explore the mechanism of corresponding antigen protein in disease development.And thus find appropriate marker for ankylosing spondylitis and possible therapeutic targets.ObjectivesPart 1: To screen autoantibodies in the plasma of patients with ankylosing spondylitis and expand samples to verify the expression of Kaiso protein autoantibodies in the plasma of ankylosing spondylitis.Part 2: To investigate the effect of Kaiso on osteogenic differentiation in vitro and in vivo.Part 3: To explore the mechanism of Kaiso’s regulation on the osteogenic differentiation.MethodsPart 1:(1)Screening the plasma of AS patients and normal control: After chip closure,chip hybridization,chip detection,data extraction and analysis,we screened out the autoantibodies specific in patients with ankylosing spondylitis.(2)Validation of plasma autoantibodies: based on the Meso Scale Discovery(MSD)technique,a sandwich method was used.First,the recombinant protein was coated on the plate bottom,and then plasma sample containing the antibody or a positive control antibody was added.After incubated for a while,sulfo-labeled secondary antibody was added.Ultimately,detected the signal based on electrochemical luminescence theory and came out with the level of antibody in different plasma samples.Part 2:(1)Establishment of Kaiso overexpression and knockdown stable cell lines: The lentivirus-infected stable cell lines were constructed through the construction of viral vectors,lentivirus packaging,cells infected by lentivirus and puromycin screening.Fluorescent quantitative PCR and Western blotting were used to verify the overexpression and knockdown efficiency.(2)Induction and detection of osteogenic differentiation: osteogenic differentiation was induced by adding 50 μg / ml ascorbic acid,10 mM sodium β-glycerophosphate and 50 ng / ml BMP2 recombinant protein in culture medium.The changes of ossification related molecules were detected by PCR at different time points.The alkaline phosphatase expression was detected by BCIP/NBT staining.Alkaline phosphatase activity was detected by alkaline phosphatase activity assay kit.Alizarin Red S staining was used to observe the formation of calcium nodules.All these were used to evaluate the osteogenic differentiation ability of different cell lines.(3)Ectopic osteogenesis experiment in nude mice: Kaiso overexpression,knockdown and corresponding control cell lines were inoculated onto the β-TCP scaffolds.The cellmaterial complexes were implanted into nude mice intramuscularly to simulate living environment.Finally,the osteogenic capacity of different stable cell lines was evaluated by HE staining and bone mineral density(BMD)detected by μCT.Part 3:(1)RNA sequencing was used to explore the changes of gene expression in Kaiso overexpression and knockdown cell lines: by RNA extraction,RNA quality testing,the establishment of cDNA library,on-machine testing,sequencing analysis and other steps to detect the differentially expressed genes in Kaiso overexpression and knockdown cell lines.And further GO,KEGG analysis was used to explore which biological processes and pathways the differential genes enrichment to.(2)PI3K-Akt signaling pathway inhibitors were added and induced by osteogenic induction medium to investigate whether this signaling pathway is involved in the process of osteogenic differentiation mediated by Kaiso.(3)The prediction and validation of the target gene that Kaiso binding: according to the bioinformatics analysis,the target gene that may be bound by the transcription factor Kaiso was predicted.ChIP-PCR and luciferase reporter gene experiments verified the binding of Kaiso to the Itga10 promoter region.(4)Itga10 knockdown assay further confirmed the relationship between Itga10 and PI3K-Akt signaling pathway,moreover the relationship between Itga10 and osteogenic differentiation.ResultsPart 1:(1)Screening the plasma of AS patients and normal control by the HuProt array.The results showed that MYLK,ASAP,SUGT1,BCL7 A,EIF2C1,KAISO,IGHG17 were existed specific in plasma of AS patients.(2)Expand the sample size to verify the existence of Kaiso protein autoantibody using the MSD technology.The results showed that the Kaiso autoantibody in early AS patients was significantly higher than normal control.The level in advanced AS patients was non-significant differences compared to normal control.Part 2:(1)The Kaiso overexpression and knockdown MC3T3-E1 stable cell lines were successfully constructed by lentivirus infection.Osteogenic induction of Kaiso overexpression and knock-down stable cell lines was carried out using osteogenic induction medium.The results showed that the expression of ossification related molecule,alkaline phosphatase activity and calcium nodule formation were significantly inhibited after Kaiso overexpression,but significantly increased after Kaiso knockdown.(2)Ectopic osteogenesis experiments in nude mice confirmed that the in vivo osteogenic capability of Kaiso-overexpressed stable cell lines was lower than that of the control,while the osteogenic potential of Kaiso-knockdown metastatic cell lines was significantly increased compared with the control.Part 3: GO analysis of the differential genes obtained by RNA sequencing showed that down-regulated genes after Kaiso overexpression and genes upregulated after Kaiso knockdown can be enriched to ossification related biological processes.KEGG analysis found that the PI3K-Akt signaling pathway was inhibited after Kaiso overexpression and was activated after Kaiso knockdown.The change of Itga10 is consistent with the change of PI3K-Akt signaling pathway.(2)PI3K-Akt signaling pathway inhibitor confirmed that this pathway is involved in Kaiso-mediated osteogenic differentiation.(3)ChIP-PCR and luciferase reporter assay showed that Kaiso inhibited the expression of Itga10 by binding to the specific sequence present in the Itga10 promoter region.(4)Itga10 knockdown experiments confirmed that Itga10 expression was positively correlated with PI3K-Akt signaling pathway,knockdown of Itga10 could inhibit osteoblast differentiation.Ectopic osteogenesis in nude mice further confirmed the inhibitory effect of Itga10 knockdown on the osteogenic differentiation.ConclusionThis study revealed that the autoantibody to the Kaiso protein was increased in early stage of ankylosing spondylitis.To further explore the relationship between the Kaiso and ossification of ankylosing spondylitis,we found that the Kaiso could regulate the PI3 KAkt signaling pathway by combining to Itga10 promoter region,thus inhibiting the process of osteogenesis differentiation.This provides a new possibility for early diagnosis and ossification intervention for AS.