RNA sequencing reveals a regulatory network of circRNA-miRNA-mRNA ceRNA in Wilms tumor

Abstract

Objective To identify the potential circRNA-miRNA-mRNA regulatory networks in Wilms tumor (WT) based on high-throughput RNA sequencing technology and bioinformatics analysis. Methods A total of 25 WT specimens were collected from the patients undergoing surgical treatment in our department during June 2021 and March 2022. The differential expression profiles of circRNA (DEcircRNA) and mRNA (DEmRNA) of 4 paired tumor and adjacent normal tissues were analyzed by RNA sequencing. Then, these differential molecules were subjected to miRanda-based miRNA target prediction to establish a complete circRNA-miRNA-mRNA regulatory network based on identical loci and co-expression. Finally, the function of the key circRNAs was validated. Results We screened 314 DEcircRNAs and 1 612 DEmRNAs out. Of these, 12 circRNAs were randomly selected by RT-qPCR in the tumor tissues to make expression verification, and 7 of them showed a trend consistent with the sequencing results. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses suggested that the parental genes of DEcircRNAs and DEmRNAs were mainly enriched in cell proliferation, cancer, and various metabolism-related pathways. The miRanda prediction tool identified common miRNA targets of DEcircRNAs and DEmRNAs as a means to construct a complete circRNA-miRNA-mRNA network. Gene set enrichment analysis (GSEA) suggested that the target genes in the ceRNA network were associated with cell cycle and immune response. To identify key downstream targets, a protein-protein interaction (PPI) network of 127 target genes in the ceRNA network was established based on the STRING database, and 10 hub genes were further identified. Among them, 4 hub genes (TP53, KANK3, LLGL1 and TOPA3) were confirmed to be closely associated with the prognosis of WT. Subsequently, a regulatory sub-network of circRNA action was constructed based on the key target genes with prognostic significance. Finally, silencing of circRNA (hsa_circ_0009035) in the sub-network was found to significantly inhibit the proliferation, migration, invasion and cell cycle distribution of WT cells by CCK-8 assay, cell scratch assay, Transwell assay and flow cytometry, respectively (P < 0.05). Conclusion The comprehensive expression of circRNAs in WT is revealed and a potential circRNA-associated ceRNA network is constructed, which profoundly clarify the regulatory mechanism of the occurrence and development of WT.

Keywords

• wilms tumor
• nephroblastoma
• rna sequencing
• circrna
• cerna network