An Exploration Of Key Signaling Pathways And Hub Genes Involved In Endometriosis Based On Whole-exome Sequencing And Gene Expression Profiling

Objective:To further understand the pathogenesis of endometriosis,to explore the key signaling pathways and hub genes involved in the development of endometriosis for new clinical diagnostic markers and therapeutic targets.Methods:Totally 26 specimens of eutopic endometrium(Eu),ectopic endometrium(Ec)and peripheral blood(PB)from women with endometriosis and control endometrium(Ctrl)from women without endometriosis were collected from clinic.The DNA of all specimens were extracted respectively for whole-exome sequencing(WES).Quality control,comparison,correction,annotation and screening of somatic mutations were performed on the raw sequencing data.Gene expression profiles of 61 Eu,Ec and Ctrl specimens were obtained from the Gene Expression Omnibus(GEO).Filtration,supplementation,standardization,logarithmetics,annotation and elimination of inter-batch variations were performed on the raw expression profiles.Multiple bioinformatics methods,including weighted gene co-expression network analysis(WGCNA),differentially expression gene analysis,enrichment analysis,protein-protein interaction(PPI)network analysis,functional module analysis and subnetwork analysis,were used to conduct an in-depth analysis of the preprocessed WES data and gene expression profiles for identification of key signaling pathways and hub genes involved in endometriosis.Results:In the section of whole-exome sequencing,data with high quality,sequencing depth and target region coverage rate were obtained,based.on which the distribution in genome,functional classification and pathogenicity of single nucleotide variant(SNV)and insertion/deletion(Indel)as well as SNV mutation spectrum were analyzed.Totally 258 eutopic endometrium-specific mutations and 768 ectopic endometrium-specific mutations were screened out,mainly enriched in signaling pathways associated with genetic information transmission,embryonic organ development,cancer,apoptosis and autophagy,for example mTOR signaling pathway.The protein-protein interaction(PPI)network was constructed,from which 8 functional modules were identified and 67 hub genes such as POLR2D,PAXIP1 and NCOA6 were singled out.In the section of gene expression profiling,weighted gene co-expression network analysis(WGCNA)was used to analyze the combined data of 3 kinds of specimens including Eu,Ec and Ctrl.Two modules,paleturquoise and darkmagenta,with high-positive correlation with Ec were screened out.Genes in the modules were mainly enriched in signaling pathways correlated with tumor and neurogenesis,for example PI3K-Akt signaling pathway.8 hub genes were identified from the paleturquoise module:LAMB2,LRP1,ANTXR1,HTRA1,FMOD,NID2,JAM3 and MYH11.6 hub genes were identified from the darkmagenta module:NFASC,ROBO3,NTRK2,NCAM1,PRELP and CHL1.Then the 3 kinds of specimens were compared in pairs,respectively 225(Ec vs Eu),131(Eu vs Ctrl)and 242(Ec vs Ctrl)differentially expressed genes(DEGs)were singled out and mainly enriched in signaling pathways associated with inflammation,immunity,migration,invasion,proliferation and embryonic development,for example Wnt signaling pathway.Three PPI networks were constructed based on the three groups of DEGs,from which 10 functional modules were identified and 67 hub genes such as ANXA1,CXCL12 and PBK were screened out.Conclusion:Endometriosis is a complex multifactorial disease.Multiple factors including inflammation,immunity,embryonic residues,apoptosis,autophagy and neurogenesis as well as migration,invasion,adhesion and proliferation abilities of eutopic endometrium may be involved in the development of Endometriosis.Key signaling pathways such as PI3K-Akt,mTOR and Wit signaling pathways and hub genes such as POLR2D,NFASC and ANXA1 may play important roles in the pathogenesis of endometriosis and related pain.Moreover,these findings can be potential diagnostic markers and therapeutic targets.