Screening Differential Proteins Based On TMT Proteomics And Functional Study Of OLFM4 In Inflammation Of RA-FLS

Objective: 1.Identification of differentially expressed proteins in synovial tissue of knee joint of rheumatoid arthritis(RA)and osteoarthritis(OA)based on TMT proteomics technology,and screening potential markers for diagnosis and treatment of RA.2.To investigate the biological function and molecular mechanism of Olfactomedin-domain family 4(OLFM4)in the development of rheumatoid arthritis synovial fibroblasts(RA-FLS).Method: 1.Identification and screening of differentially expressed proteins in knee synovial tissues of patients with OA and RA by TMT proteomics technology.Through GO function enrichment and KEGG pathway enrichment analysis,combined with sequencing information in GEO datebase database,using GEO 2R algorithm to identify target genes.2.Collect synovial tissue of knee joint from patients with OA and RA,and verify the expression level of OLFM4 in synovial tissue by WB,RT-PCR and IHC techniques.3.The type and location of target gene-expressing cells were detected by immuno-British-light co-localization technique.DAPI staining was used to nucleate the target gene.Vimentin was used as a cell marker of RA-FLS and OLFM4 was used as the target gene.4.Synovial fibroblasts were extracted from synovial tissues of patients with OA and RA and cultured for passage 3-6.TNF-alpha,IL-1 and LPS were used to stimulate synovial fibroblasts.IL-6 level was used as an indicator of inflammatory activation.WB and RT-q PCR were used to detect the expression level of target genes.5.At the time point of RA-FLS stimulated by TNF-alpha,the expression level of target gene was detected by RT-q PCR.6.Designing and synthesizing small interfering RNA(si RNA)and negative control RNA(NC)of the target gene OLFM4,transfecting si-RNA into RA-FLS with the help of lip2000 transfection reagent,detecting the silencing efficiency of OLFM4 by WB and RT-q PCR,and detecting the expression level of CXCL-9,CXCL-10,CXCL-11 and MMP-1 genes in inflammation pathway by RT-q PCR.7.Silencing the target gene in RA-FLS by si-OLFM4 and replenishing it with TNF-alpha cytokines 24 hours later,using IL-6 as the standard,RT-q PCR was used to detect the effect of target gene replenishment and the changes of CXCL-9,CXCL-10,CXCL-11 and MMP-1 inflammatory factors in TNF-alpha inflammation pathway,and to analyze the function and role of target gene in inflammation.8.OLFM4 was silenced in RA-FLS stimulated by TNF-alpha.Cell proliferation was measured by flow cytometry at 72 hours.Results: 1.In this study,TMT relative quantitative proteomics was used to screen differential proteins in synovial tissues of OA and RA patients.A total of 4822 differentially expressed proteins were identified in synovial tissues.A total of 510 differentially expressed proteins were screened according to the criteria of 1.2-fold expression(up-regulation is greater than 1.2-fold or down-regulation is less than 0.83-fold)and Pvalue < 0.05.In RA-vs-OA group,239 differential proteins were up-regulated and 271 differential proteins were down-regulated.Through the analysis of GO function and KEGG pathway,it was found that the functions of these differentially expressed proteins were mainly concentrated in binding,catalytic activity,structural molecular activity,molecular function regulator and transporter activity.The differentially expressed proteins were mainly involved in response to stimulus and other important biological processes.The candidate gene OLFM4 was identified through the GO functionalenrichment,KEGG pathway enrichment and differential multiple of differential proteins.2.Synovial tissues were collected from knee arthroplasty patients in the joint surgery department of Xi’an Honghui Hospital.WB and RT-q PCR were used to verify the expression of OLFM4 in RA,including protein level,RNA level and immunohistochemistry.The results showed that OLFM4 expression in RA was significantly higher than that in OA,and there were statistical differences.3.Fibroblast synovial cells of OA and RA were extracted and cultured for 3-6 generations.TNF-alpha(10ng/ml)was added to stimulate both RA-FLS and OA-FLS.Sample proteins and RNA were collected.The results of RT-q PCR and Western Blot assay showed that the expression level of OLFM4 in RA-FLS was significantly higher than that in OA-FLS(vs control,P < 0).In addition,CXCL-9,CXCL-10,CXCL11,MMP-1,IL-6 and other related inflammatory factors in TNF-alpha inflammation pathway increased.4.Total RNA was extracted 24 hours after transfection of si-OLFM4 into RA-FLS.RT-q PCR was used to detect the total RNA.The results showed that the silencing efficiency of si-OLFM4 in RA-FLS was higher than 80%(si OLFM4-vs-si NC).5.Silicon-OLFM4 was transfected into RA-FLS cells to inhibit the expression of target genes.TNF-alpha was added 24 hours later to stimulate synovial cells.RT-q PCR results showed that the expression of CXCL-9,CXCL-10,CXCL11,MMP-1,IL-6 and other related inflammatory factors in TNF-alpha inflammation pathway showed the same trend in OLFM4 replenishment experiment.6.OLFM4 was silenced under TNF-alpha-stimulated RA-FLS condition.After 72 hours,the cell proliferation was detected by flow cytometry,indicating that OLFM4 could Promoting cell proliferation in RA-FLS.Conclusion: In this paper,the differentially expressed gene OLFM4 was screened out by TMT quantitative proteomics in OA and RA tissues,and the expression of OLFM4 in RA was higher than that in OA.In addition,cell experiments showed that the expression ofOLFM4 in RA-FLS was significantly higher than that in OA-FLS stimulated by cytokine TNF-alpha,and the expression of MMP-1,CXCL9,CXCL11 and IL-6 in inflammation pathway increased,thus activating inflammation and promoting the occurrence and development of inflammation.OLFM4 could also inhibit the proliferation of RA-FLS,which provided a theoretical basis for the occurrence and development of chronic inflammation in RA.