Research On Differential Methylated Genes In Synovium Of Rheumatoid Arthritis Based On Methylation Microarray Technique

Research backgroundRheumatoid arthritis(RA),a systemic autoimmune disease with the main clinical manifestation characterized by multi-joint symmetric aggressive arthritis,can lead to joint deformity and loss of function at the late stage of disease development,which brings heavy burden to patients and their families.Therefore,seeking for more effective therapeutic targets is essential to prevent joint destruction in RA patients,thereby improving the quality of life of patients.So far,the etiology of RA has not been clear,environmental and genetic factors have traditionally been considered as the main causes of RA.In recent years,as an emerging subject,epigenetics has been proved to be involved in the pathogenesis of RA by more and more studies,among which DNA methylation is an important epigenetic modification.In this study,we adopted the latest Illumina methylation 850 k chip technology to compare and conduct genome-wide DNA methylation analysis in synovium of RA patients and osteoarthritis(OA)patients,and screen out the differentially methylated genes(DMGs),explore the expression levels of DMGs,further elaborated the role of DNA methylation plays in the pathogenesis of RA.Part One: Research on DNA methylation of synovium in rheumatoid arthritis based on methylation microarrayObjective: To investigate the role of DNA methylation in synovitis and find new therapeutic targets for RA.Methods: 7 RA inpatients and 9 age-and gender-matched OA inpatients from inpatients who underwent total knee arthroplasty(TKA)between September 2017 and November 2019 at the General Hospital of Western Theater Command(Chengdu,China)were enrolled in this study.The average ages of RA and OA patients were 57.0y(52.0,65.0y)and 61.0y(55.5,66.5y),respectively,without significant difference(P=0.314).After obtaining the informed consent of the patient,the discarded synovium samples were obtained to extract DNA postoperatively.DNA quality was measured using the Qubit ds DNA HS Assay Kit.For each qualified sample,DNA was converted by bisulfite using the Zymo EZ DNA Methylation kit,and bisulfite-converted DNA subsequently underwent amplification,incubation,fragmentation,precipitation,and resuspension.Then,resuspended DNA was hybridized to the Infinium Methylation EPIC Bead Chip microarray(850k),after the arrays were washed,expanded,dyed,they were scanned in Illumina i Scan,the scanner captured fluorescence emitted by fluorescence groups and produced high-resolution images stored as the resulting raw data(IDATs).R package(version 3.1.1)was used to control raw data quality,read methylation levels,conduct differential analysis,and screen out the DMGs with P<0.05.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were undertaken using cluster Profiler,plotting showed the results of enrichment with most significance.The STRING database(version 11.0)was utilized to measure interactions between identified DMGs.Results: Through comprehensive analysis and quality control,a total of 76,514 DML were screened out.Among them,51,859 hypermethylated DML were distributed in 29,277 hypermethylated DMGs,and 24,655 hypomethylated DML were distributed in 16,484 hypomethylated DMGs,respectively.67.78% of DML were hypermethylated in RA.The heatmap showed 79.30% of DML were hypermethylated among the top 1000 DML.The distribution location of DML was analyzed,39.20% of DML were located in the gene body,32.36% of DML were located in the intergenic region,and 9.20% of DML were located in the5’UTR.In terms of GO enrichment,DML were mainly enriched in the cell morphogenesis of neuronal differentiation(P=7.00E-31),axon development(P=1.20E-27),chemical synaptic transmission regulation(P=6.60E-27)and other biological processes.In terms of cell structure,they were mainly enriched in pre-synapse(P=1.24E-11),synaptic membrane(P=1.89E-12),glutamate energetic synapse(P=4.14E-09).In terms of cell function,they were mainly enriched in actin binding(P=1.81E-14),GTP-enzyme binding(P=1.71E-12),matrix specific channel activity(P=3.10E-12)and other functions.KEGG pathway analysis showed that DMGs were enriched in axon guidance(P=2.20E-11),calcium signaling pathway(P=3.20E-12),RAP1 signaling pathway(P=8.90E-13)and other pathways.DMGs related to axon guidance were demonstrated to interact with each other through exploration with STRING.Part two: Expression of semaphorins in synovium of rheumatoid arthritis and its clinical valueObjective: To detect the expressions of semaphorin3C(SEMA3C)and semaphorin3F(SEMA3F)in synovium of rheumatoid arthritis(RA),and analyze the correlations with clinical inflammatory indicators,and clarify its clinical value in evaluating the disease progression of RA patients.Method: Synovium specimens of knee joints from 8 patients with RA and 8 patients with OA were selected,using immunohistochemistry to observe and contrast the distribution of SEMA3 C,SEMA3F and tyrosine hydrogenase(TH)in synovium of RA and OA,western blotting and real-time quantitative polymerase chain reaction(RT-q PCR)were used to contrast the expression levels of SEMA3 C and SEMA3 F between RA synovium and OA synovium,the correlation relationships between expression levels of SEMA3 C,SEMA3F and erythrocyte sedimentation rate(ESR),anti-cyclic citrullinated peptide antibodies(ACPA),rheumatoid factor(RF),C-reactive protein(CRP),platelet distribution width(PDW),mean platelet volume(MPV),platelet aggregation(PCT)and platelet count(PLT)were analyzed by Pearson method.Results: SEMA3 C and SEMA3 F were more widely distributed in RA synovium than those in OA synovium;TH was more widely distributed in OA synovium than that in RA synovium.The expression levels of protein and m RNA of SEMA3 C and SEMA3 F in RA synovium were higher than those in OA synovium(P<0.05).Protein expression level of SEMA3 C was negatively correlated with MPV(r=-0.842,P<0.001)and positively correlated with RF(r=0.727,P=0.007);protein expression level of SEMA3 F was positively correlated with ESR(r=0.821,P=0.002)and negatively correlated with PDW(r=-0.707,P=0.009).Conclusion1.67.78% of DML showed hypermethylation in RA.2.DMGs were enriched in neuronal differentiation and axon development,synaptic transmission and axonogenesis,as well as several cell components related to synapses,axon guidance pathway.3.SEMA3 C and SEMA3 F were highly expressed in RA synovium,and they were correlated with clinical inflammatory indicators.