Screening And Identification Of Biomarkers For Systemic Sclerosis Via Microarray Technology

ObjectiveTo screen the hub genes related to systemic sclerosis(SSc)and investigate their biological functions and potential roles underlying SSc,a variety of bioinformatics analysis tools were applied so as to provide possible mechanism of this disease from molecular biology point of view and ground work for making progress of the diagnosis and prognosis of SSc.Materials and Methods(1)Screening of differentially expressed genes(DEGs):two microarray datasets,GSE95065 and GSE76885,were downloaded from the GEO(Gene Expression Omnibus)database and intra-group data repeatability tests were conducted using Pearson's correlation test and principal component analysis(PCA).DEGs were identified using an online tool,GEO2R.The screening criteria were P<0.05,|log₂FC|?1.5.(2)Functional enrichment analysis of DEGs:Gene Ontology(GO)and Kyotoencyclopedia of genes and genomes(KEGG)were performed to analyze the function and to observe the pathways enrichment of DEGs via DAVID online analysis website.(3)Interaction analysis of proteins encoded by DEGs and screening of hub genes:protein-protein interaction(PPI)network was constructed via STRING online database and visualized by software Cytoscape.Plugin MCODE loaded in the software was then used to construct functional modules and further filter out the top10 DEGs considered as key hub genes of SSc.(4)Functional analysis and diagnostic experiment of hub genes:GO enrichment analysis and KEGG biological pathway enrichment analysis were carried out on hub genes,and the receiver operator character(ROC)curve analysis was performed to determine the abilities of these hub genes for predicting SSc.Results(1)DEGs screening results:according to the screening criteria,compared with the Con group 106 DEGs in common with the two datasets were obtained.(2)Results of GO enrichment analysis:DEGs were significantly enriched in the biological processes(BP)including inflammatory responses,extracellular matrix organization,positive regulation of cell proliferation,innate immune response,defense response to virus,positive regulation of cell migration,osteoblast differentiation and its positive regulation,platelet degranulation,etc;cellular components(CC)were mainly in the extracellular regions,including extracellular exosomes,extracellular region,extracellular space,extracellular matrix,cell surface and cell-cell junctions,intracellular components including intracellular space,perinuclear region of the cytoplasm,Golgi apparatus,Golgi cisternae membranes,coat protein I-coated vesicles and host cell;molecular function of DEGs were mainly enriched in terms such as protein binding,receptor binding,GTPase activity and transcription regulatory region,etc.(3)KEGG signaling pathway enrichment analysis of DEGs:DEGs were mainly involved in pathways related to cytokine-cytokine receptor interaction,salmonella infection and legionellosis.(4)10 key hub genes and their further analysis results:CLU,SOCS3,PRSS23,BMP4,TLR4,CYR61,IL6,CALU,TNC and SCG2.Hub genes were significantly enriched in the BP including regulation of apoptotic processes,inflammatory responses,positive regulation of extracellular signal regulated kinase(ERK1)and ERK2 cascade,positive regulation of NF?B transcription factor activity;CC includes extracellular space and extracellular matrix;MF of hub genes were mainly enriched in cytokine activity and chemokine activity.KEGG signaling pathway enrichment analysis showed that hub genes were mainly involved in pi3k-akt signaling pathway and influenza infection.The expression of all hub genes was associated with the diagnosis of SSc(0.7<area under the curve(AUC)<1;P?0.05).Conclusions(1)This study using rigorous bioinformatics methods integrated two datasets from the GEO database and screened out 106 DEGs and 10 hub genes(CLU,SOCS3PRSS23,BMP4,TLR4,CYR61,IL6,CALU,TNC and SCG2)which were associated with SSc.(2)In the biological processes DEGs are mainly involved in inflammation and innate immune responses,cell proliferation and migration,extracellular matrix synthesis,virus defense reaction;KEGG pathways mainly enriched in the interaction of cytokines and their receptors,salmonella infection and legionellosis.These results indicated that infection is most likely to be involved in the pathogenesis of SSc.(3)The role of TLR4 in the progression of fibrosis remains to be elucidated;the expression of IL6 was up-regulated in SSc,and it has become a hot potential therapeutic target for SSc;this study is the first to propose that CALU is low expressed in SSc.How CALU participates in the pathogenesis of SSc needs further studies to gain insights.