Identification of potential circRNAs and circRNA-miRNA-mRNA regulatory network in the development of diabetic foot ulcers by integrated bioinformatics analysis

Abstract

We aimed to explore the mechanism of circular RNAs (circRNAs) and provide potential biomarkers for molecular therapy of diabetic foot ulcers (DFU). Gene expression profile of GSE114248, including five normal samples and five DFU samples, was downloaded from GEO database. Differentially expressed circRNAs (DEcircRNAs) between two groups were identified. Then, DEcircRNA-miRNA and miRNA-mRNA interaction was revealed, followed by the circRNA-miRNA-mRNA network construction. Moreover, functional and pathway analysis were performed based on mRNAs, followed by the DM-related pathway exploration. Specific binding sites for key circRNAs and associated miRNAs were under investigation. Finally, RT-qPCR was used to verify the candidate the relative expression level of circRNA between normal tissues and DFU. Totally, 65 DEcircRNAs were revealed between two groups, followed by 113 circRNA-miRNA-mRNA interactions explored. The mRNAs in these interactions were mainly assembled in functions like cell proliferation and pathways. Moreover, a total of 11 DM-related pathways were revealed. Finally, circRNA-miRNA specific binding-site analysis revealed two key circRNAs, for example, circRNA_072697 and circRNA_405463, corresponding to their miRNAs. These two circRNAs were novel biomarkers for DFU. circRNA_072697 acted as a sponge of miR-3150a-3p in the progression of DFU via regulating KRAS. MAPK signaling pathway might contribute to the development of DFU.

Keywords: circRNA-miRNA-mRNA; circRNAs; diabetic foot ulcers; function and pathway enrichment analysis; miRNA-mRNA binding sites.

FIGURE 1

The volcano plots and heatmap for differentially expressed circular RNAs (DEcircRNAs)between diabetic foot ulcers (DFU)sample and normal sample. A, the volcano plots in current study; the X‐axis represents the value of log2 fold change, while the Y‐axis represents the value of ‐log10; the top5 up‐ and down‐regulated circRNAs were shown. B, the heatmap in current study; color from green to red indicated low to high representation value

FIGURE 2

The circRNA‐miRNA‐mRNA interaction network. The red triangle represents miRNA; the green square represented circRNA; the yellow node represents the mRNA

FIGURE 3

The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. A, The result of KEGG pathway enrichment. B, The result of GO functional enrichment. The X‐axis represents the number of genes in certain item; the Y‐axis represents the different items of functions or pathways; different color indicated different miRNA

FIGURE 4

The Venn plot for pathways enriched by mRNAs and explored in Toxicogenomics Database (CTD) database. The purple circle represented the pathways enriched by target mRNAs of miRNAs. The yellow circle represented the DM‐associated pathways explored in CTD database

FIGURE 5

The detail information of the binding sites for circRNA_072697‐miR‐3150a‐3p and circRNA_405463‐miR‐4649‐3p. The “|” indicated perfect match of binding site; the “:” indicated incomplete match of the binding site

FIGURE 6

Relative expression level of circRNA_072697 in normal tissues (control) and DFU. DFU represented diabetic foot ulcers. *, P < .05. GAPDH was used as the reference gene