Integrated bioinformatics analysis for the identification of hub genes and signaling pathways related to circANRIL

Background. Antisense noncoding RNA in the INK4 locus (ANRIL) is located on human chromosome 9p21, and modulation of ANRIL expression mediates susceptibility to some important human disease, including atherosclerosis (AS) and tumors, by affecting the cell cycle circANRIL and linear ANRIL are isoforms of ANRIL. However, it remains unclear whether these isoforms have distinct functions. In our research, we constructed a circANRIL overexpression plasmid, transfected it into HEK-293T cell line, and explored potential core genes and signaling pathways related to the important differential mechanisms between the circANRIL-overexpressing cell line and control cells through bioinformatics analysis. Methods. Stable circANRIL-overexpressing (circANRIL-OE) HEK-293T cells and control cells were generated by infection with the circANRIL-OE lentiviral vector or a negative control vector, and successful transfection was confirmed by conventional flurescence microscopy and quantitative real-time PCR (qRT-PCR). Next, differentially expressed genes (DEGs) between circANRIL-OE cells and control cells were detected. Subsequently, Gene Ontology (GO) biological process (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to explore the principal functions of the significant DEGs. A protein-protein interaction (PPI) network and competing endogenous RNA (ceRNA) network were constructed in Cytoscape to determine circularRNA (circRNA)- microRNA(miRNA)-messenger RNA (mRNA) interactions and hub genes, and qRT-PCR was used to verify changes in the expression of these identified target genes. Results. The successful construction of circANRIL-OE cells was confirmed by plasmid sequencing, visualization with fluorescence microscopy and qRT-PCR. A total of 1745 DEGs between the circANRIL-OE group and control were identified, GO BP analysis showed that these genes were mostly related to RNA biosynthesis and processing, regulation of transcription and signal transduction. The KEGG pathway analysis showed that the up regulated DEGs were mainly enriched in the MAPK signaling pathway. Five associated target genes were identified in the ceRNA network and biological function analyses. The mRNA levels of these five genes and ANRIL were detected by qRT-PCR, but only COL5A2 and WDR3 showed significantly different expression in circANRIL-OE cells.