Ribavirin Modulates Keloid Fibroblast Proliferation,Motility And Extracellular Matrix Production Through Targeting EIF4E/eIF4Gl Mediated Epithelial-Mesenchymal Transition(EMT)

BackgroundKeloid is a benign fibroproliferative disease,which leads to abnormal dermal fibrosis due to excessive deposition of extracellular matrix(ECM)[1,1].Although it is considered a benign tumor,keloids resemble malignant tumors in the stages of hyperplasia,invasion and anti-apoptosis[3].Epithelial mesenchymal transition(EMT)is an important mechanism for regulating tumor metastasis behavior.Therefore,the identification of EMT biomarkers is essential for a comprehensive understanding of the pathogenesis of keloids.At present,the pathogenesis of keloid remains unclear,and treatment methods are limited.There is still a recurrence rate after surgical resection and radiotherapy.Therefore,it is urgent to understand its pathogenesis and accelerate the development of therapeutic methods.In order to further optimize the treatment of keloid,explore its pathogenesis and improve the cure rate is the key research of this subject.In this project,the following two aspects of bioinformatics retrieval and analysis and in vitro experiment were studied:Part Ⅰ:The biomarkers of key genes associated with epithelial-mesenchymal transition in human keloids.ObjectivesThe epithelial interstitial transition(EMT)phenomenon was identified as an important mechanism of fibrosis disease metastasis.Understanding the biomarkers of epithelial interstitial transition(EMT)in keloids is important for resolving the pathogenesis of keloid and its relationship to treatment.MethodsThe differentially expressed genes(DEGs)GSE92566 dataset were identified and downloaded from GEO databases,with 3 normal skin and 4 keloid tissues.Furthermore,EMTrelated genes were downloaded from dbEMT 2.0 databases and intersected with GSE92566 DEGs to identify EMT-related-DEGs(ERDEGs).Subsequently,the ERDEGs were used for Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes,(KEGG),Gene Set Enrichment Analysis(GSEA),Protein-protein interaction(PPI)and miRNAsmRNAs network analysis.The ERDEGs were imported to cMAP databases,whereas hub genes were imported to DGidb databases,in order to predict small molecules for EMT inhibition.Final step was carried out via qRT-PCR to validate the findings.ResultsA total of 122 ERDEGs were identified,including 59 upregulated and 63 down-regulated genes.Moreover,enrichment analysis revealed that focal adhesion,AMPK signal pathway,Wnt signal pathway,and EMT biological process were significantly enriched.STRING databases and Cytoscape software were used to construct the PPI network and EMT-related hub genes.Further,3 modules were explored from the PPI network using the Molecular Complex Detection(MCODE)plugin.In the Cytohubba plugin,10 hub genes were explored,including FN1,EGF,SOX9,CDH2,PROM1,EPCAM,KRT19,ITGB1,CD24,and KRT18.These genes were then enriched for the focal adhesion pathway.We constructed a microRNA(miRNA)-mRNA network,which predicted hsa-miR-155-5p(8 edges),hsa-miR-124-3p(7 edges),hsa-miR-145-5p(5 edges),hsa-miR-20a-5p(5 edges)and hsa-let-7b-5p(4 edges)as the most connected miRNAs regulating EMT.Based on the ERDEGs and 10 hub genes mentioned above,ribavirin demonstrated high drug-targeting relevance.Finally,qRT-PCR confirmed that the expression of FN1,ITGB1,CDH2,and EPCAMcorroborated with previous findings.ConclusionIn summary,this bioinformatic work provides novel EMT biomarkers in keloids and predicts new small target molecules for keloid therapy.These findings improve the understanding of keloid pathogenesis,providing new treatment options.Part Ⅱ:Role of ribavirin in keloid fibroblast function.ObjectivesThis study aimed to clarify the potential of applying the ribavirin in keloid treatment by elucidating its functional roles and mechanisms in fibroblasts.MethodsWe treated human keloid fibroblasts(HKFs)with different concentrations of ribavirin,and performed cell morphology observation and toxicity assessment.CCK-8 assay was used to detect cell viability,EdU assay was to detect cell proliferation,flow cytometry was to detect cell cycle and apoptosis,wound healing assay was to detect wound healing in vitro,transwell assay was to detect cell invasion and migration,and qPCR/western blot was to detect extracellular matrix(ECM)and epithelial-mesenchymal transition(EMT)-related parameters.ResultsRibavirin evidently inhibited HKF proliferation,migration and invasion,induced its G0/G1 arrest and apoptosis.Moreover,ribavirin suppressed ECM production as well as EMT in HKFs.eIF4E and eIF4GI were highly expressed in HKFs,which were significantly downregulated by ribavirin treatment.Silencing eIF4E or eIF4GI evidently inhibited cell proliferation,migration,invasion,EMT and ECM production.Further,overexpression of eIF4E or eIF4GI partially reversed the effects of ribavirin on above HKF functions.ConclusionRibavirin could regulate keloid fibroblast proliferation,motility,and ECM production by targeting eIF4E/eIF4GI-mediated EMT,which may be a possible treatment option in clinical keloid diagnosis and treatment.