Screening of potential biomarkers in propofol-induced neurotoxicity via bioinformatics prediction and experimental verification

Abstract

Objectives: To identify hub genes and biological processes of propofol-induced neurotoxicity and promote the development of pediatric anesthesiology.

Methods: We downloaded the GSE106799 dataset from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were screened, then Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and Gene Set Enrichment analyses were performed on all DEGs. We identified potential ferroptosis genes in the pathogenesis of propofol-induced neurotoxicity. A key module was obtained after performing weighted gene co-expression network analysis (WGCNA) on the GSE106799 dataset. Hub genes were identified after the least absolute shrinkage and selection operator (LASSO) regression analysis of the intersection of DEGs and genes from the key module. We established a competing endogenous RNA network and predicted potential drugs according to the hub genes. Total RNA and proteins were extracted for real-time quantitative polymerase chain reaction and Western blotting, respectively.

Results: A total of 112 DEGs, including 76 upregulated and 36 downregulated ones were screened out. Propofol-induced neurotoxicity involved processes such as nervous system development, activation of JAK/STAT and MAPK signaling pathways, vascular regeneration, and oxidative stress. The results of WGCNA suggested that the tan module was the most strongly associated with propofol-induced neurotoxicity. We identified 4 hub genes (EGR4, HAO1, ITK and GM14446) after LASSO regression analysis. Animal experiments demonstrated that propofol caused overexpression of the protein levels of HAO1, ITK and inflammatory factors in the brain, as well as the mRNA levels of HAO1, ITK and GM14446. Propofol inhibited expression of EGR4 at mRNA and protein levels.

Conclusions: Previous studies have demonstrated that EGR4, HAO1, ITK and GM14446 play a role in intellectual development, neuroinflammation and neuronal differentiation. These hub genes may help us to find new preventive and therapeutic targets for propofol-induced neurotoxicity.

Keywords: Propofol; neurotoxicity; potential biomarkers.

Figure 1

Study workflow.

Figure 2

DEGs and enrichment analysis. A. Volcano plot of DEGs. B. Heatmap of DEGs. Red represents high expression, and blue represents low expression. C-F. Enrichment analysis: GO, KEGG and GSEA. DEDs: Differentially Expressed Genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA: Gene Set Enrichment Analysis.

Figure 3

Identification of key module and genes via WGCNA. A. Clustering dendrogram of samples with trait heatmap. B. Analysis of network topology for various soft-thresholding powers. C. Clustering dendrogram of genes, with dissimilarity based on topological overlap, together with assigned module colors. D. Module-trait associations: each row corresponds to a module eigengene and each column to a trait. Each cell contains the corresponding correlation and P value. E. MM and GS scatter plots for the key module tan. The vertical line is |MM| = 0.8 and the horizontal line is |GS| = 0.8. The key genes of the module are in the box in the upper right corner. WGCNA: Weighted Gene Co-expression Network Analysis; GS: Gene Significance; MM: Module Membership.

Figure 4

Screening of hub genes. A. Venn diagram of key module genes and DEGs. B. The most proper log λ value in the LASSO model. C. The log λ value of the DEGs related to propofol-induced neurotoxicity in the LASSO model. D-G. The areas under the ROC curve of 4 hub genes were all 1.0. DEDs: Differentially Expressed Genes; LASSO: Least Absolute Shrinkage and Selection Operator; ROC: Receiver Operating Characteristic.

Figure 5

Heatmap of ferroptosis-related differentially expressed genes.

Figure 6

ceRNA network related to 4 hub genes. Orange ovals indicate hub genes, purple triangles indicate lncRNAs, and green diamonds indicate miRNAs.

Figure 7

Drug-hub gene network. The red octagons represent hub genes, and purple diamonds represent drugs.

Figure 8

Expression levels of hub genes. A-D. mRNA expression levels of hub genes in the GSE106799 dataset. E. Expression levels of hub genes in experimental sample. *P<0.05, **P<0.01, ***P<0.001.

Figure 9

Western blotting validation of the expression levels of inflammatory factors and hub genes. A. Western blotting of NF-κB, IL-6, HAO1, ITK and EGR4. *P<0.05. B. Analysis of western bloting.