| Background:Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease characterized by proliferation of fibroblast-like synoviocytes(FLSs),infiltration of immune cells and pannus formation.The progression of the disease can result in damage to the synovial tissue of the joint,leading to eventual teratogenesis and disability.But up to now,the pathogenesis of RA has not been fully explained.As an important participant in synovial inflammation,the role of FLSs in the occurrence and development of disease has attracted more and more attention.Fibroblast activating proteinα(FAPα)is mainly expressed on the surface of activated fibroblasts.Studies have shown that high expression of FAPαis associated with increased expression in tumor,pulmonary fibrosis and other diseases,and is associated with invasion.Therefore,the role of FAPαin RA-FLSs remains to be further studied and discussed.Objective:(1)Through the analysis of public sequencing data,we screened the differential genes related to the expression of FAPαin synovial tissue of RA patients and healthy controls(HC)to predict the pathways through which FAPαmight affect the progression of RA disease.(2)In vitro cell experiments were conducted to investigate the effects of FAPαon the proliferation,cycle,apoptosis,migration and invasion ability of RA-FLSs,and whether the effects of FAPαon cell function were related to SHH signaling pathway,so as to reveal the possible biological functions of FAPαin RA-FLSs,and provide experimental basis for the clinical transformation of targeting FAPα~+FLSs in the treatment of RA in the future.Methods:1.Bioinformatics analysis:(1)The synovial tissue and platform data of RA and HC were downloaded from NCBI GEO database.To the original data after standardizing,using R language"limma"package of RA and HC group differences in gene screening,set a threshold value for|log FC|>1 and P<0.05;According to gene sequencing data in FAPαexpression in two groups data can be divided into high and low expression,gene screening differences,set a threshold value for|log FC|>1 and P<0.05.The intersection of two groups of differential genes was selected as the key gene for the next analysis.The"ggplot2"package in R language is used to draw volcanic maps and cluster heat maps of RA and HC.(2)Go analysis and KEGG pathway analysis of key genes were conducted by using R package cluster Profiler(10)in Bioconductor,and P<0.05 was statistically significant.(3)The STRING database was used to construct the protein-protein interaction network,and the possible protein interactions among key genes were analyzed to find the possible disease-related genes of FAPα.2.Extraction and identification of FLSs:(1)Both RA-FLSs and FLSs of osteoarthritis(OA)were isolated from synovial tissue of patients undergoing joint replacement under aseptic conditions.The tissues were cut up and digested with trypsin,and then cultured in complete medium containing DMEM.FLSs were adherent cells and grew slowly.After 3-4 days,adherent cells reached more than 80%,and subculture was performed.The cells used in this study were all 3-6 generation cells.(2)Cell identification was mainly carried out by observing cell morphology,using FLSs surface markers(positive)FAPα,PDPN and CDH11,and macrophage surface marker(negative)CD68.3.si RNA transfection experiments:Design and synthesis of FAPαwith small interfering RNA(si RNA).si RNA is transfected into RA-FLSs using GP-Transfect-Mate.The Blank control group(Blank group),negative control group(NC group)and FAPαinterference group(si RNA group)were set.Real-time PCR and Western blot were used to detect the inhibition of FAPαfrom m RNA and protein levels,and si RNA with the highest interference rate was screened for subsequent experiments.4.Detection methods of cell proliferation,cycle,apoptosis,migration and invasion ability:(1)CCK8 assay was used to detect the proliferation activity of the experimental group and the control group.Flow cytometry was used to detect the cell cycle and apoptosis.(2)The protein expressions of SHH,PTCH1 and GLI1 in SHH signaling pathway were detected by Western blot.Results:1.The role of FAPαin RA synovial by bioinformatics analysis:through the HC and RA synovial tissue genetic data analysis,identified in FAPαkey genes related to express,through the GO analysis and KEGG analysis found that the main enrichment of key genes in rheumatoid arthritis,white blood cell migration,cell associated with the connection between cells and extracellular matrix collagen,receptor ligands and cytokine activity,protein coupled receptor receptors,cytokines,chemokines and metal peptide enzyme activity,CCR and CXCR chemokine receptors,protein fiber connection combination etc.Several important signaling pathways.The hub genes MMP1,MMP3,MMP13,CXCL9 and CXCL13 related to the expression of FAPαwere screened by protein-protein interaction network.Real-time PCR was used to verify the RA-FLSs samples,and it was found that the expression of MMP3,MMP13 and FAPαwere correlated.2.Effects of FAPαon FLSs cell function:(1)Influence on cell proliferation and cycle:cell proliferation test showed that compared with the NC group,the cell growth rate of the si RNA group was inhibited 24h and 48h after transfection,with statistical difference(P<0.05).Cell cycle test showed that the proportion of G0/G1 phase and S phase in si RNA group was not significantly changed compared with NC group,but the proportion of FLSs in G2/M phase was decreased(P<0.05).(2)Effect on apoptosis:apoptosis test showed that there was no significant change in apoptosis between the si RNA group and the NC group 48h after transfection,and the difference was not statistically significant(P>0.05).(3)The effect on cell migration and invasion ability:compared with the NC group,the migration ability of the si RNA group at 24h after transfection showed no significant difference(P>0.05),while the invasion experiment showed that the invasion ability of the si RNA group at 24h after transfection was significantly reduced,with a statistically significant difference(P<0.05).(4)Influence on SHH signaling pathway in RA-FLSs:Western blot results showed that the relative expression levels of SHH and GLI1 in si RNA group were decreased compared with NC group(P<0.05),but PTCH1 expression had no significant difference(P>0.05).Conclusion:1.The analysis of gene sequencing data showed that FAPαcould affect the synovial pathological changes of RA by affecting cell functions and signaling pathways such as cytokine receptor binding,chemokine activity and metallopeptidase activity,and was related to core genes such as MMP3 and MMP13.2.The increased expression of FAPαcan promote the proliferation and invasion ability of RA-FLSs,but has no obvious effect on cell apoptosis.3.The effect of FAPαon the proliferation and invasion ability of RA-FLSs was related to the activation of SHH signaling pathway. |
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