Roles And Mechanism Of Follistatin-like Protein 1 And Its Regulatory Factors In Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a multifactorial systemic autoimmune disease of unknown etiology,associated with a chronic inflammatory process,which primarily affects the joints and extra-articular tissues,including the heart,lungs,digestive system,skin,and nervous system.RA is mainly characterized by morning stiffness and joint pain,which may destroy the affected joints as the disease progresses,leading to disability and a reduced quality of life in severe cases.Studies have shown that RA is a global disease occurring to any race,ethnic group,nationality and age,etc.and that there are gender differences in the prevalence of RA,which is 3-5 times higher in women than in men.In recent years,the progress in clinical diagnosis and treatment of RA have improved the clinical conditions of patients,but the specific pathogenesis of RA is not clear and there are still some difficulties in reducing disease activity and effectively preventing complications,which impose a heavy burden on patients and society.Studies suggest that the interaction of environmental,genetic and stochastic factors may underlie the complexity of the biomolecular mechanisms of rheumatoid arthritis.Studies have reported that the inflammatory response can occur before the onset of arthritic symptoms(the so-called pre-rheumatoid arthritis),resulting from interactions between genetic modifications in genomic structure and environmental factors that lead to denaturation of autoantigens,such as immunoglobulin G(IgG),type 2 collagen and wave proteins,which are not recognized by the autoimmune system as self-structured and are taken up by antigen-presenting cells(APCs),activating CD4+helper T cells and the immune response through interactive signaling between B cells and T cells.Synovial fibroblasts play an important role in the disease progression of rheumatoid arthritis by continuous proliferation and formation of granulations.SFs are produced by bone marrow mesenchymal cells.In RA,SFs manifest excessive proliferation,reduced apoptosis,enhanced cell migration and invasion,which invade the extracellular matrix and aggravate joint damage.In addition,SFs produce and release a variety of inflammatory cytokines,chemokines,and matrix degrading molecules that cause chronic synovitis in joints,produce large amounts of fluid,and further erode and destroy articular cartilage at a later stage.The expression of interferon regulatory factor 1 was significantly increased in Synovial fibroblasts in the synovial layer and sublayers of RA patients,and it plays an important role in TNF-driven RA inflammation.However,the mechanisms of SFs acting in the pathogenesis and progression of RA are still unclear.Therefore,SFs in RA can be a potential and safe therapeutic target,and exploring SFs-associated factors involved in the progression of RA and their gene regulatory networks is considered a research priority for RA to develop novel and effective therapeutic strategies.Follistatin-like protein 1(FSTL1)is an extracellular matrix glycoprotein widely distributed in various organs throughout the body and is highly expressed in myocardial infarction and ischemia-reperfusion injury.Under ischemic conditions,FSTL1 may promote endothelial cell and vascular regeneration by affecting the endothelial Protein Kinase B-Nitric Oxide Synthase signaling pathway.It has been found that FSTL1 can induce spontaneous inflammation.Overexpression of FSTL1 can promote IL-6 secretion by SFs,stimulate macrophages and induce the release of pro-inflammatory factors in joints.FSTL1 overexpression is involved in cell proliferation,migration and invasiveness of SFs to the extracellular matrix by promoting TLR4 and NF-κB expression in the development and progression of RA.Another study showed that knockdown or antagonism of FSTL1 could bring about effective anti-fibrosis and reduce the severity of arthritis.However,it is still unclear how FSTL1 and its gene regulatory factors plays their roles in the development and progression of RA.Therefore,the analysis of FSTL1-related molecules such as transcription factors(TFs)and gene regulatory networks that regulate FSTL1 can help further understand the molecular mechanism of RA and provide a new theoretical basis for the diagnosis and treatment of the disease.Long non-coding RNA(LncRNA),longer than 200bp,involved in cell proliferation and differentiation.ZNFX1 antisense RNA 1(ZFAS1)is a new type of lncRNA,located on chromosome 20q13.13,which is the antisense RNA of ZNFX1.ZFAS1 was found to be highly expressed in SFs of RA patients,and silent ZFAS1 inhibited the migration and invasion of RA-SFs.This study aimed to investigate the potential roles of SFs in the development of RA and the important regulon associated with FSTL1 in it,as well as the interaction between lncRNA ZFAS1 and FSTL1 so as to search for potential therapeutic targets and provide a theoretical basis for targeted treatment of RA.To clearly articulate the above issues,this paper will address the following two aspects:Part Ⅰ.Single-cell transcriptome-based analysis of FSTL1 in RA-SFsObjectives:This experiment aimed to investigate the potential roles of SFs and the predominant regulators(regulons)associated with FSTL1 in the development of RA so as to find potential therapeutic targets and provide a theoretical basis for targeted treatment of rheumatoid arthritis.Methods:1.The transcript data of SFs were obtained by searching the GEO database(http://www.ncbi.nih.nlm.gov/geo)and processed for screening,normalization,and quality control.2.Principal Component Analysis(PCA)named "RunPCA"function was applied to linearly transform the single-cell transcript data to extract the principal components.3.The Uniform Manifold Approximation and Projection(UMAP)technique was used to downscale and cluster cells and identify subsets of SFs.4.Single-cell Regulatory Network Inference And Clustering(SCENIC)was used to analyze and compute key transcriptional regulatory networks in these SF subgroups.5.Extraction application pySCENIC(v.0.11)was used to further explore the functional differences of the SF subgroups and mine the regulatory module where FSTL1 is extracted for analysis of transcriptional regulatory status.6.The STRING database(https://cn.string-db.org/)was applied to make predictions and construct the Protein-Protein Interaction(PPI)network.7.Differential expression analysis of pseudo-bulk transcript data of RA and OA was performed by applying "DESeq2".8.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis and Gene Ontology(GO)enrichment analysis were adopted to analyze signaling pathways.9.The possible functions of IRF1 and FSTL1 in RA were investigated by calculating the respective enriched gene sets of IRF1 and FSTL1 by using GSEA analysis.Results:1.A total of three SF subsets were identified from the single cell transcripts of SFs and SF subset 3 was the most important in RA.2.Among the three SFs subset,FSTL1 and IRF1 are mainly enriched in SF-3 subset.3.IRF1 was upregulated in RA,which is the main regulatory factor in RA gene regulatory network.FSTL1 is regulated by IRF1.Conclusions:1.SF-3 subset in RA-SFs was the key pathogenic cell subset in the progress of RA.2.The expression of FSTL1 is increased in SF-3 subset,which is a key factor in gene regulation network.3.FSTL1 was regulated by IRF1,and FSTL1 affected the progression of RA through the regulation by IRF 1Part Ⅱ.Effects of FSTL1 and its regulatory factor ZFAS1 on proliferation,apoptosis and inflammatory reaction of RA-SFs in RAObjectives:This experiment was to further investigate the mechanism of FSTL1 in RA and to explore the relationship and regulatory mechanism between FSTL1 and ZFAS1 so as to provide a new theoretical basis for the molecular mechanism of RA.Methods:1.A rat model of RA was constructed and changes in the expression of FSTL1 in the synovial tissue and SFs of the model were detected by qRT-PCR and western.2.The FSTL1 downexpression or overexpression plasmid was transfected into MH7A cells,and cell proliferation of MH7A was detected by CCK-8,and apoptosisrelated molecules was detected by qRT-PCR and western blot.3.The correlation between FSTL1 and ZFAS1 was analyzed by biobanking,the expression of ZFAS1 in synovial tissue and SFs in the animal model were detected by qRT-PCR and western blot,and the correlation between FSTL1 and ZFAS1 expression in the animal model was analyzed.4.The ZFAS1 overexpression plasmid was transfected into MH7A cells,and the proliferation of MH7A cells was detected by CCK-8,and the apoptosis of MH7A cells was detected by Western Blot.5.qRT-PCR was applied to detect the expression of inflammatory cytokines after FSTL1 silencing,overexpression and ZFAS1 overexpression transfection,and finally Western Blot was used to detect the expression of inflammatory cytokines.Results:1.The expression of FSTL1 in both synovial tissue and SFs increased in CIA rats.2.Down-regulation of FSTL1 expression can inhibit the proliferation of MH7A cells,while overexpression of FSTL1 promoted cell proliferation.3.Down-regulation of FSTL1 promoted apoptosis and the expression of Caspase-3 and Poly ADP-ribose Polymerase;overexpression of FSTL1 inhibited apoptosis and suppressed the expression of caspase-3 and Poly ADP-ribose Polymerase.4.The expression of FSTL1 and ZFAS1 in motion system was positively correlated by biochemical analysis.The expression of ZFAS1 was elevated in synovial tissues and SFs and there was a positive correlation between and the expression of FSTL1 and ZFAS1.5.Overexpression of ZFAS1 promoted cell proliferation and reduces scilencing FSTL1-induced apoptosis to a certain extent.6.qRT-PCR results showed that downregulating the expression of FSTL1 inhibited the expression of IL-6 and TNF-α;Overexpression of FSTL1 promoted the expression of IL-6 and TNF-α;Overexpression of ZFAS1 promoted the expression of IL-6 and TNF-α and m itigate to some degree the reduced expression of IL-6 and TNFa cause by down-regulation of FSTL1.Conclusions:1.The expression of FSTL1 and ZFAS1 in synovial tissue and SFs increased and there was a positive correlation between the expression of FSTL1 and ZFAS1.2.Through the regulation of ZFAS1 expression,FSTL1 could promote cell proliferation,inhibit cell apoptosis,promote the expression of inflammatory cytokines so as to contribute to the inflammatory reaction of RA.