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Updated: Sep 25, 2025
In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions
Published on: October 21, 2022
Integrated bioinformatics analysis for the identification of hub genes and signaling pathways related to circANRIL
Qiuyan Qin1, Pengfei Zheng2, Ronghui Tu3
1Department of Neurology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China.
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.
Insights
Circular ANRIL (circANRIL) overexpression impacts gene expression and signaling pathways. This study identified COL5A2 and WDR3 as differentially expressed genes, suggesting distinct functions for circANRIL isoforms.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Antisense noncoding RNA in the INK4 locus (ANRIL) is implicated in diseases like atherosclerosis and cancer.
- ANRIL exists as circular (circANRIL) and linear isoforms, but their distinct functions are not well understood.
Purpose of the Study:
- To investigate the functional differences between circANRIL and linear ANRIL.
- To identify core genes and signaling pathways affected by circANRIL overexpression.
Main Methods:
- Overexpression of circANRIL in HEK-293T cells using lentiviral vectors.
- Differential gene expression analysis (DEGs) via RNA sequencing.
- Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses.
- Construction of protein-protein interaction (PPI) and competing endogenous RNA (ceRNA) networks.
- Validation of target gene expression using quantitative real-time PCR (qRT-PCR).
Main Results:
- 1745 differentially expressed genes (DEGs) were identified between circANRIL-overexpressing and control cells.
- GO analysis indicated DEGs are involved in RNA biosynthesis, processing, transcription regulation, and signal transduction.
- KEGG analysis revealed enrichment in the MAPK signaling pathway.
- Five target genes were identified, with COL5A2 and WDR3 showing significantly altered expression.
Conclusions:
- circANRIL overexpression significantly alters gene expression profiles and impacts the MAPK signaling pathway.
- COL5A2 and WDR3 are potential downstream targets of circANRIL, suggesting distinct functional roles for circANRIL isoforms.
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