Analysis Of LncRNA-associated CeRNA Network In IgAN

BackgroundIgA nephropathy(IgAN)is the most common primary glomerular disease in young people in the world.It is particularly common in Asian populations.About 40%of patients can develop into end-stage renal disease(ESRD)within 20 years,requiring renal replacement therapy.At present,the diagnosis and risk stratification of adult IgAN mainly depend on invasive renal biopsy.Therefore,further clarifying the molecular mechanism of IgAN is helpful for the diagnosis and targeted therapy of IgAN.Competing endogenous RNAs(ceRNAs)can regulate gene expression by competing binding microRNAs(miRNAs).A ceRNA regulatory network shows long non-coding RNA(lncRNA),miRNA and mRNA levels.A large number of evidences shows that ceRNAs are involved in many biological processes in organisms and play an important role in them,and can lead to the occurrence and development of various human kidney diseases,but the role of ceRNAs in IgAN is less studied.ObjectivesIn this study,the differentially expressed genes in renal tissues of IgAN patients were detected and analyzed by RNA-sequencing(RNA-Seq),and the lncRNA-associated ceRNA network was constructed to explore the potential role of these ceRNA molecules in IgAN.Methods1.We collected renal tissues from 6 IgAN patients and 3 controls,and used RNA-seq technology to sequence lncRNA,mRNA and miRNA.2.Gene ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed according to the obtained differentially expressed genes.3.The online database was used to predict the targeted binding of differentially expressed genes and construct the ceRNA network.4.In order to verify the sequencing results,another 6 renal tissues(3 cases of IgAN and 3 cases of control group)were used to detect 9 differentially expressed genes randomly selected by quantitative reverse transcription(qRT-PCR).5.The dual luciferase reporter assay was performed to verify the targeted binding of miR-199a-5p to fibroblast growth factor 11(FGF11)in ceRNA network.Results1.Compared with the control group,there were 562 significantly different lncRNAs,1993 significantly different mRNAs and 58 significantly different miRNAs in renal tissues of IgAN patients.2.The results of GO function and KEGG pathway enrichment analysis showed that the significantly different mRNAs were mainly enriched in leukocyte migration,regulation of lymphocyte activation,interaction between cells and cytokine receptors,and chemokine signaling pathways.3.We predicted the target genes of differentially expressed genes,and obtained 90 pairs of differentially expressed lncRNA-miRNAs and 652 pairs of differentially expressed miRNA-mRNAs.The IncRNA-associated ceRNA network was successfully constructed,including 24 differentially expressed miRNAs,66 differentially expressed lncRNAs and 435 differentially expressed mRNAs.4.Three IncRNAs,three miRNAs and three mRNAs were detected by real-time fluorescent quantitative PCR,which were consistent with the sequencing results.5.The dual luciferase reporter assay verified the targeted binding between miR-199a-5p and FGF11.ConclusionsIn summary,we detected differentially expressed lncRNAs,miRNAs and mRNAs associated with IgAN by whole transcriptome sequencing,and successfully constructed lncRNA-associated ceRNA networks.These new networks may be potential biomarkers or therapeutic targets in IgAN.