Microarray Analysis Identifies Key Differentially Expressed Circular RNAs in Aged Mice With Postoperative Cognitive Dysfunction

Abstract

Postoperative cognitive dysfunction (POCD) is a common complication in elderly patients. Circular RNAs (circRNAs) may contribute to neurodegenerative diseases. However, the role of circRNAs in POCD in aged mice has not yet been reported. This study aimed to explore the potential circRNAs in a POCD model. First, a circRNA microarray was used to analyze the expression profiles. Differentially expressed circRNAs were validated using quantitative real-time polymerase chain reaction. A bioinformatics analysis was then used to construct a competing endogenous RNA (ceRNA) network. The database for annotation, visualization, and integrated discovery was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of circRNA-related genes. Moreover, protein-protein interactions were analyzed to predict the circRNA-regulated hub genes using the STRING and molecular complex detection plug-in of Cytoscape. Microarray screen 124 predicted circRNAs in the POCD of aged mice. We found that the up/downregulated circRNAs were involved in multiple signaling pathways. Hub genes, including Egfr and Prkacb, were identified and may be regulated by ceRNA networks. These results suggest that circRNAs are dysexpressed in the hippocampus and may contribute to POCD in aged mice.

Keywords: ceRNA network; circRNAs; expression profile; miRNAs; postoperative cognitive dysfunction.

Figure 1

Hippocampus-dependent learning and memory, but not hippocampus-independent learning and memory or motor activity was impaired by anesthesia/surgery in aged mice. (A) The experimental process’ schematic diagram. (B) The baseline of total distance the elderly mice traveled in open field test (OFT) during the training period 1 day before surgery. (C) The baseline of freezing time (%) in fear conditioning test (FCT) of aged mice during the training duration 1 day before surgery. (D) The total distance traveled in OFT 3 days after the operation. (E) Freezing time percentage in the FCT context test 3 days after surgery. (F) Freezing time percentage in the FCT tone test 3 days postoperatively. Data for each group are shown as the mean ± standard error (n = 10). **p < 0.01 compared with the control group.

Figure 2

Character of differentially expressed circRNA in the postoperative cognitive dysfunction (POCD) and control groups. (A) Distribution of circRNAs in the six samples (C1–3: control group; P1–3: POCD group). (B) The difference expression of circRNA is shown by a scatter plot between the control and POCD groups. (C) The differential circRNA expression is shown by volcano diagrams between the control and POCD groups. (D) Thirty-six downregulated and 88 upregulated circRNAs in POCD are shown in the heatmap. (E) The pie chart showed the transcriptional sources of the differentially expressed circRNAs. (F) Chromosome locations of the differentially expressed circRNAs.

Figure 3

Conformation of the six prospected circRNAs with qRT-PCR. RNAs from the POCD and control groups were involved (A–F). *p < 0.05 compared with the control group.

Figure 4

circRNA-miRNA-mRNA network analysis. (A) The ceRNA network for the three upregulated circRNAs. (B) The ceRNA network for the three downregulated circRNAs.

Figure 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. (A,B) The KEGG enrichment analysis of the up/downregulated circRNA’s predicted genes.

Figure 6

Protein-protein analysis of circRNAs regulated genes. (A,E) The protein-protein interaction network analysis for the up/downregulated genes. (B,F) Top three sub-network modules that are predicted by MCODE in the Cytoscape software. (C,G) Top 10 hub genes screened by CytohHubba in the Cytoscape software. (D,H) The mRNA expression level of Egfr and Prkacb validation. *p < 0.05 compared with the control group.