Construction Of CircRNA-miRNA-mRNA Regulatory Network Of Recurrent Implantation Failure To Explore Its Potential Pathogenesis

Objective: Circular ribonucleic acid（circRNA）plays an important role in the regulation of gene expression,but its specific role in the pathogenesis of recurrent implantation failure（RIF）remains unclear.The purpose of this study is to identify the circular RNA（circRNA）associated with RIF and explore the potential role of their regulatory network in the pathogenesis of RIF patients to provide ideas and basis for the etiological research and treatment of RIF.Methods: Microarray data of RIF related circRNA,micro RNA（miRNA）and messenger RNA（mRNA）were downloaded from Gene Expression Omnibus（GEO）database to identify differentially expressed circRNA（DEC）,miRNA（DEmi R）and mRNA（or gene,DEG）by the limma package in the Rstudio software.Overlapping miRNA was obtained by interacting predicted miRNA which was performed by the Circular RNA Interactome online tool and DEmi R,and so was corresponding circRNA.Next,target mRNA prediction of miRNA was performed by Target Scan,mi RDB and mi RTar Base online software,and the intersection mRNA was selected as the predicted mRNA using Venn diagram in the Rstudio software.Overlapping mRNA was obtained through interactively predicted mRNA and the DEG,and the circRNA-miRNA-mRNA network was constructed by Cytoscape version 3.8.0 software,then the protein-protein interaction（PPI）network was constructed by Search Tool for the Retrieval of Interacting Gene（STRING）software and the hubgene was identified by cyto Hubba plug-in,and the circRNA-miRNA-hubgene subnetwork was constructed by Cytoscape version 3.8.0 software to find the upstream circRNA and miRNA.Finally,the Gene Oncology（GO）annotation analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis of the hubgene were performed and Reactome FI plug-in was used for reactome analysis to comprehensively analyze hubgene mechanism in RIF.Results: A total of 3 RIF related microarray data（GSE147442,GSE71332 and GSE103465）were downloaded from the GEO database,and 8 upregulated DECs,5downregulated DECs,56 downregulated DEmi Rs,104 upregulated DEmi Rs,429 upregulated DEGs and 1067 downregulated DEGs were identified by the limma package.The miRNA response elements（MREs）of these 13 DECs were then predicted.7 overlapping miRNAs were obtained by intersecting the predicted miRNAs and DEmi Rs and so were corresponding 6 circRNAs.Then,56 overlapping mRNAs were identified by intersecting the predicted target mRNAs of 7 miRNAs with 1496 DEGs.The circRNA-miRNA-mRNA network was constructed by 6 circRNAs,7 miRNAs and56 mRNAs and a PPI network was constructed by 56 mRNAs and 4 hubgenes（YWHAZ,JAK2,MYH9 and RAP2C）were identified by cyto Hubba plug-in.The circRNA-miRNA-hubgene subnetwork with 3 circRNAs（hsa_circ₀058161,hsa_circ₀030162 and hsa_circ₀033392）,3 miRNAs（hsa-mi R-1290,hsa-mi R-375 and hsa-mi R-1305）and 4 hubgenes was constructed by Cytoscape version 3.8.0software,comprising 9 regulatory modules,namely hsa_circ₀058161/hsa-mi R-1290/YWHAZ regulatory axis,hsa_circ₀058161/hsa-mi R-1290/RAP2 C regulatory axis,hsa_circ₀030162/hsa-mi R-375/JAK2 regulatory axis,hsa_circ₀030162/hsa-mi R-375/YWHAZ regulatory axis,hsa_circ₀033392/hsa-mi R-375/JAK2 regulatory axis,hsa_circ₀033392/hsa-mi R-375/YWHAZ regulatory axis,hsa_circ₀033392/hsa-mi R-1305/YWHAZ regulatory axis,hsa_circ₀033392/hsa-mi R-1305/MYH9 regulatory axis and hsa_circ₀033392/hsa-mi R-1305/RAP2 C regulatory axis.Functional enrichment analysis identified that these four hubgenes were mainly involved in the regulation of “actin filament organization”,“focal adhesion”,“cadherin binding” and “tight junction” pathways in the biological process of RIF and reactome analysis identified 4 hubgenes mainly involved in “RHO GTPases activate PKNs”,“interleukin-3（IL-3）,IL-5 and granulocyte macrophage colony stimulating factor（GM-CSF）signaling” and “glucose transporter 4（GLUT4）to the plasma membrane”reactome pathway.Conclusion: In this study,DECs,DEmi Rs and DEGs were screened out and a circRNA-associated ce RNA network was constructed.The circRNA-miRNA-hubgene regulatory subnetwork revealed that the key circRNAs were hsa＿circ＿0058161,hsa＿circ＿0030162 and hsa＿circ＿0033392 which may be involved in the occurrence and development and main biological processes of RIF patients including “actin filament organization”,“focal adhesion”,“cadherin binding”,“tight junctions”,“RHO GTPases activate PKNs”,“IL-3,IL-5 and GM-CSF signaling” and “GLUT4 to the plasma membrane ” by YWHAZ,JAK2,MYH9 and RAP2C gene.