Comprehensive analysis of circRNA-miRNA-mRNA networks to reveal potential cell death, inflammation and oxidative stress-related targets for postoperative cognitive dysfunction.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a common neurological complication involving the hippocampus, a brain region critical for learning and memory. Circular RNA (circRNA) plays an important role in regulating gene expression. Our objective is to predict and verify a circRNA-miRNA-mRNA network in POCD. METHODS: Microarray data of POCD mouse models were retrieved from the Gene Expression Omnibus (GEO) database. Differential expression analysis was used to identify differentially expressed circRNA, miRNA, and mRNA. An in silico circRNA-miRNA-mRNA network was subsequently predicted based on miRNA-mRNA pairs and miRNA-circRNA pairs obtained from public databases. From this global network, three hub subnetworks based on the competing endogenous RNA (ceRNA) hypothesis were analyzed. In hippocampal tissue of POCD mice, RT-qPCR quantified key circRNAs, miRNAs, and mRNAs, with protein levels of the corresponding mRNA confirmed by western blot. Dual-luciferase assays in HT22 cells verified interactions between mmu_circ_0001838 (mmu_circRNA_009748) and mmu-miR-337-3p, and between mmu-miR-337-3p and PLA2G12B mRNA. RESULTS: Analysis of publicly datasets related to POCD identified 12 genes potentially associated with programmed cell death, inflammation, immunity, and oxidative stress in POCD. According to these results, we successfully predicted a circRNA-miRNA-mRNA network, comprising 4 circRNAs, 14 miRNAs and 12 mRNAs. Furthermore, three circRNA-miRNA-mRNA subnetworks were then derived based on the ceRNA hypothesis, which included 3 circRNAs, 4 miRNAs and 8 mRNAs: mmu_circRNA_016232-mmu_miR-6912-5p-four mRNAs (GBP2, BCL3, ADGRL4, PLN), mmu_circRNA_009748-mmu_miR-362-3p/mmu_miR-337-3p-two mRNAs (RSAD2, PLA2G12B), and mmu_circRNA_009396-mmu_miR-7030-5p-two mRNAs (SLC25A13, GPR171). The AUC value of each predicted circRNA-miRNA-mRNA path in diagnosing POCD exceeded 0.8. RT-qPCR results validated that the levels of mmu_circRNA_016232, mmu_circRNA_009748, mmu_miR-7030-5p, GBP2 mRNA, BCL3 mRNA, ADGRL4 mRNA, PLN mRNA, RSAD2 mRNA and PLA2G12B mRNA were elevated, while the levels of mmu_circRNA_009396, mmu_miR-6912-5p, mmu_miR-362-3p, mmu_miR-337-3p, SLC25A13 mRNA, and GPR171 mRNA were reduced in the hippocampal tissues of POCD mice compared to the control group. Western blot corroborated the protein-level changes for the corresponding mRNAs. Dual-luciferase assays further confirmed the direct interactions of mmu_circ_0001838 with mmu-miR-337-3p and mmu-miR-337-3p with PLA2G12B mRNA. CONCLUSION: This study predicted and verified three critical circRNA-miRNA-mRNA regulatory axes, providing new insights into the molecular mechanisms underlying POCD and identifying potential biomarkers for its diagnosis and intervention strategies.