Analysis Of MRNA And MiRNA Differential Expression Profiles Related To Urethral Injury In Rats Based On High-Throughput Sequencing

Objective:Through bioinformatics analysis,the genomic expression profiles of mRNA and miRNA in urethral injury tissue were studied to explore the potential promoter factors associated with inflammation,tissue damage and repair to urethral injury.The key genes and potential regulatory miRNAs were screened out to provide ideas and directions for the further study of the mechanism of urethral injury.Methods:1.Four male SD rats were randomly divided into injured group and normal group,with two in each group.The urethral injury model was established by scalding the urethral mucosa of rats in the injured group with a hot iron rod.After 3 days of modeling,the rats in the injured group and the normal group were killed respectively,and the urethral tissue was removed.2.The urethral tissues of the injured group and the normal group were sent to the sequencing company forRNA and smallRNA sequencing.3.Transcriptome analysis was performed to identify differentially expressed mRNA between the injured group and the normal group.GO functional enrichment analysis,KEGG pathway enrichment analysis and GSEA enrichment analysis were performed for the differentially expressed mRNAs.4.By smallRNA analysis,differentially expressed miRNAs were found between the injured group and the normal group.The target genes of differentially expressed miRNA were identified by Hit Sensor algorithm,and the GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for the target genes.Results:1.Results of mRNA differential expression gene analysis(1)Through transcriptome analysis,a total of 166 differentially expressed mRNAs were identified between the injured group and the normal group(FDR < 0.05),of which69 were upregulated and 97 were downregulated.(2)GO functional enrichment analysis describes the biological process,functional localization information and functions of gene products from three aspects: biological process(BP),cell component(CC)and molecular function(MF).The GO terms of upregulated DEGs were mainly involved in negative regulation of cytosolic calcium ion concentration,positive regulation of epithelial cell differentiation,inner cell mass cell proliferation and other biological processes,extracellular space,stress fiber and other cell components,as well as the molecular functions of actin filament binding,peptidase activator activity and protein binding.On the other hand,the GO terms of downregulated DEGs were mainly involved in negative regulation of endopeptidase activity,muscle contraction,antimicrobial humoral immune response mediated by antimicrobial peptide and other biological processes,kinocilium,collagen-containing extracellular matrix and other cell components,as well as the molecular functions of endopeptidase inhibitor activity,small molecule binding and WW domain binding.(3)KEGG pathway enrichment analysis can be used to identify metabolic and signaling pathways involved in specific genomes.The upregulated DEGs were mainly involved in Cell adhesion molecules(CAMs),c GMP-PKG signaling pathway,ECMreceptor interaction,Phagosome,Cellular senescence and other signaling pathways.However,the downregulated DEGs were mainly associated with Tight junction,Endocytosis,Graft-versus-host disease and other signaling pathways.(4)GSEA enrichment analysis showed that protein activation cascade,complement activation,complement and coagulation cascades,positive regulation of vascular endothelial growth factor production,chemokine-mediated signaling pathway and monocyte chemotaxis gene sets were closely related to phenotype injured.However,oxidative phosphorylation,mitochondrial protein complex and respiratory electron transport chain gene sets were closely related to phenotype normal.(5)Eight genes related to urethral injury were screened out in this study,including Ccl6,Casp1,LOC100911413,C4 b,Rnf7,Mylk,Col14a1 and Igfbp3.They were significantly upregulated in urethral injury tissues,and functional annotation showed that they were involved in inflammation and immune response(Ccl6,Casp1,LOC100911413,C4 b,Rnf7),tissue repair(Mylk,LOC100911413,Col14a1),apoptosis(Casp1,Igfbp3)and other processes.2.Analysis results of miRNA differentially expressed genes(1)By smallRNA analysis,a total of 10 differentially expressed miRNAs were found between the injured group and the normal group(P < 0.05),among which 6 were upregulated and 4 were downregulated.(2)Through the Hit Sensor algorithm,6 upregulated miRNAs found a total of 59 target genes,and 4 downregulated miRNAs found a total of 23 target genes.(3)The GO terms of miRNA target genes showed that the target genes of the differentially expressed upregulated miRNAs were mainly involved in vascular endothelial growth factor receptor-2 signaling pathway,positive chemotaxis,tube formation,regulation of p38 MAPK cascade,epithelial cell differentiation and other biological processes,cytoplasm,cytosol and other cell components,as well as the molecular functions of vascular endothelial growth factor receptor 2 binding,plateletderived growth factor receptor binding and protein tyrosine phosphatase activity.On the other hand,the target genes of the differentially expressed downregulated miRNAs were mainly associated with regulation of mRNA stability,multicellular organismal reproductive process,positive regulation of cell adhesion molecule production and other biological processes,protein-containing complex,nucleus and other cell components,as well as the molecular functions of mRNA 3’-UTR binding and c AMP response element binding protein binding.(4)KEGG pathway showed that the target genes of the differentially expressed upregulated miRNAs were significantly enriched in PI3K-Akt signaling pathway,Apoptosis,Cell adhesion molecules(CAMs)and other signaling pathways.However,the target genes of the differentially expressed downregulated miRNAs were significantly enriched inRNA transport,Notch signaling pathway,Metabolic pathways,HIf-1 signaling pathway and other signaling pathways.(5)Through smallRNA analysis,potential regulatory miRNAs that may be related to urethral injury were screened out as mir-339-5p and mir-31a-5p with upregulation,mir-486 with downregulation.Conclusion:1.Ccl6,Casp1,LOC100911413,C4 b,Rnf7,Mylk,Col14a1 and Igfbp3 were significantly upregulated in urethral injury tissues,which was expected to become an intervention target for the treatment of urethral injury.2.mir-339-5p,mir-31a-5p and mir-486 may become diagnostic biomarkers and therapeutic intervention targets for early urethral injury.