Ferroptosis-related gene MAPK3 is associated with the neurological outcome after cardiac arrest

Abstract

Background: Neuronal ferroptosis is closely related to the disease of the nervous system, and the objective of the present study was to recognize and verify the potential ferroptosis-related genes to forecast the neurological outcome after cardiac arrest.

Methods: Cardiac Arrest-related microarray datasets GSE29540 and GSE92696 were downloaded from GEO and batch normalization of the expression data was performed using "sva" of the R package. GSE29540 was analyzed to identify DEGs. Venn diagram was applied to recognize ferroptosis-related DEGs from the DEGs. Subsequently, The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed, and PPI network was applied to screen hub genes. Receiver operating characteristic (ROC) curves were adopted to determine the predictive value of the biomarkers, and the GSE92696 dataset was applied to further evaluate the diagnostic efficacy of the biomarkers. We explore transcription factors and miRNAs associated with hub genes. The "CIBERSORT" package of R was utilized to analyse the proportion infiltrating immune cells. Finally, validated by a series of experiments at the cellular level.

Results: 112 overlapping ferroptosis-related DEGs were further obtained via intersecting these DEGs and ferroptosis-related genes. The GO and KEGG analysis demonstrate that ferroptosis-related DEGs are mainly involved in response to oxidative stress, ferroptosis, apoptosis, IL-17 signalling pathway, autophagy, toll-like receptor signalling pathway. The top 10 hub genes were selected, including HIF1A, MAPK3, PPARA, IL1B, PTGS2, RELA, TLR4, KEAP1, SREBF1, SIRT6. Only MAPK3 was upregulated in both GSE29540 and GAE92696. The AUC values of the MAPK3 are 0.654 and 0.850 in GSE29540 and GSE92696 respectively. The result of miRNAs associated with hub genes indicates that hsa-miR-214-3p and hsa-miR-483-5p can regulate the expression of MAPK3. MAPK3 was positively correlated with naive B cells, macrophages M0, activated dendritic cells and negatively correlated with activated CD4 memory T cells, CD8 T cells, and memory B cells. Compared to the OGD4/R24 group, the OGD4/R12 group had higher MAPK3 expression at both mRNA and protein levels and more severe ferroptosis.

Conclusion: In summary, the MAPK3 ferroptosis-related gene could be used as a biomarker to predict the neurological outcome after cardiac arrest. Potential biological pathways provide novel insights into the pathogenesis of cardiac arrest.

Fig 1. Flowchart.

Fig 2. DEGs between good outcome and poor outcome of neurological function.

(A) The heatmap shows top 50 upregulated and downregulated DEGs. The X-axis delegates the sample type, and the Y-axis delegates the DEGs. (B) Volcano plot exhibit DEGs,the red points show upregulated genes, and the green points represent downregulated genes. The X-axis delegates the logFC, and the Y-axis represents the -log10 (P.Value). P values < 0.05 was identified as significant differences. (C) Venn diagram show ferroptosis differentially expressed genes. Ferroptosis differentially expressed genes were selected by intersecting ferroptosis dataset with GSE29540 DEGs.

Fig 3. GO and KEGG enrichment analyses of 112 ferroptosis-related DEGs.

The (A) bar plot and (B) bubble of Significant GO analysis. The (C) bar plot and (D) bubble show Significant KEGG pathways.

Fig 4

(A): PPI network of ferroptosis-related DEGs. (B):The PPI networks was visualized using Cytoscape software. (C):The top10 ferroptosis-related DEGs using MCC.

Fig 5

(A-J) Expression of hub geenes in GSE92696.

Fig 6

(A) ROC curve of MAPK3 in the GSE29540; (B) ROC curve of MAPK3 in another dataset of GSE92696.

Fig 7

(A). Venn diagram show common miRNA. (B). Interactions MAPK3 and transcription factors.

Fig 8

(A). Proportions of 22 types of immune cells. (B). Heatmap visualize the correlation of the infiltration of immune cells. (C). Violin plot of the proportion of 22 types of immune cell infiltrates.

Fig 9

(A-G) Correlations between between MAPK3 and infiltrating immune cells.

Fig 10. SY5Y were hypoxic for 4 hours and reoxygenated for 12 or 24 hours to establish the OGD/R cell model.

The OGD4/R12 group of SY5Y cells was more severely damaged and had more pronounced ferroptosis than the OGD4/R24 group. A. Use the CCK8 Kit to determine cell viability. Cell viability of OGD4/R12 group and OGD4/R24 group. B. Intracellular ROS were detected using DCFH-DA fluorescence assay. ROS detected with flow cytometry. C. Confocal microscopy images and fluorescence intensity of intracellular ROS detected by DCFH-DA fluorescence assay. The scale bar represents 50 μM. D. Confocal microscopy images and JC-1 fluorescence ratio of MMPs detected by the JC-1 kit. Increased red fluorescence indicated the formation of JC-1 aggregates, suggesting a relatively intact mitochondrial membrane. Enhanced green fluorescence indicated the production of JC-1 monomer, suggesting mitochondrial membrane disruption. The scale bar represents 50 μM. E. Detection of MDA, GSH and Fe2+ levels in OGD4/R12 group and OGD4/R24 group by kit. (*p<0.05, **p<0.01, ***p<0.001 OGD4/R24 compared with OGD4/R12, In vitro experiments repeated three times).

Fig 11. SY5Y were hypoxic for 4 hours and reoxygenated for 12 or 24 hours to establish the OGD/R cell model.

Expression of the MAPK3 and ferroptosis signature genes at mRNA and protein levels in the OGD4/R12 and OGD4/R24 groups. A. The mRNA level of MAPK3、GPX4、FTH1、TFR1 were measured in the OGD4/R12 and OGD4/R24 groups by qPCR. B-E. The treated cells were incubated with primary or secondary antibodies. The expression of MAPK3/1 (b1), p-MAPK3/1 (b2), FTH1 (D), TFR1 (E) (ECD channel) and GPX4 (D) (FITC channel) protein were detected by flow cytometry. F-H. The treated cells were fixed, permeabilized, blocked and incubated with primary antibodies, secondary antibodies and probes. The expression of the MAPK3 (f1), p-MAPK3/1 (f2), FTH1 (H) (AF594) and GPX4 (G) (FITC) proteins was observed by immunofluorescence. (*p<0.05, **p<0.01, ***p<0.001 OGD4/R24 compared with OGD4/R12, In vitro experiments repeated three times).