Competing endogenous RNA network analysis of the molecular mechanisms of ischemic stroke

Abstract

Background: Ischemic stroke (IS) is a serious neurological disease that largely results in long-term disability and death. Extensive evidence has indicated that the activation of inflammation and ferroptosis significantly contribute to the development of IS pathology. However, the underlying molecular mechanism remains unclear. In this study, we aimed to identify potential biomarkers associated with IS through the construction of a competing endogenous RNA (ceRNA) network and to investigate the possible inflammatory and ferroptosis-related molecular mechanisms.

Results: We identified 178 differentially expressed target messenger RNAs (DETmRNAs) associated with IS. As revealed through enrichment analysis, the DEmRNAs were mainly enriched in the inflammatory signaling pathways and also related to ferroptosis mechanism. The CIBERSORT algorithm showed immune infiltration landscapes in which the naïve B cells, naïve T cells, and monocytes had statistically different numbers in the cerebral infarction group compared with the control group. A ceRNA network was constructed in this study involving 44 long non-coding RNAs (lncRNAs), 15 microRNAs (miRNAs), and 160 messenger RNAs (mRNAs). We used the receiver operating characteristic (ROC) analysis to identify three miRNAs (miR-103a-3p, miR-140-3p, and miR-17-5p), one mRNA (TLR4), and one lncRNA (NEAT1) as the potential key biomarkers of the ceRNA network. The key mRNA and lncRNA were shown to be highly related to the ferroptosis mechanism of IS. The expression of these key biomarkers was also further validated by a method of quantitative real-time polymerase chain reaction in SH-SY5Y cells, and the validated results were consistent with the findings predicted by bioinformatics.

Conclusion: Our results suggest that the ceRNA network may exert an important role in the inflammatory and ferroptosis molecular mechanisms of IS, providing new insight into therapeutic IS targets.

Keywords: Biomarker; Ferroptosis; Inflammatory response; Ischemic stroke; Network analysis.

Fig. 1

Flow chart of overall analysis

Fig. 2

Volcano plots: red plot represented upregulation, and blue plot represented downregulation; a GSE110993; b GSE117064；c GSE58294；d GSE198710

Fig. 3

Venn diagrams: a Screen DEmiRNAs by the intersection of GSE110993 and GSE117064; b Screen DETmRNAs by the intersection of DEmRNAs and target mRNAs; c Screen DElncRNAs by intersection of DElncRNAs and target lncRNAs

Fig. 4

IncRNAs-miRNAs-mRNAs regulatory network. Every node symbolizes one gene, and each edge indicates the interaction between genes. The shape of triangle represents IncRNAs, diamond represents miRNAs, and regular hexagon represents mRNAs. The blue color genes symbolize downregulated genes, while the red color indicates upregulated genes

Fig. 5

GO/KEGG function enrichment analysis: a biological process; b cellular component; c molecular function; d KEGG enrichment analysis (www.kegg.jp/kegg/kegg1.html)

Fig. 6

a a protein-protein interaction network; b Top 10 mRNAs ranked by Degree score; c Top 10 mRNAs ranked by MCC score; d Screen hub mRNAs by intersection of the top 10 in Degree and MCC

Fig. 7

Evaluation and visualization of immune cell infiltration: a Immune cell infiltration map of cerebral infarction group; b Immune cell infiltration map between acute cerebral infarction group and control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 8

Screen for key mRNA and lncRNA: a Screen potential ferroptosis-related mRNA by the intersection of human ferroptosis-related genes and potential key mRNAs; b NEAT1 was identified by intersection of DETlncRNAs for the 3 key miRNAs; c NEAT1 happens to be contained in human ferroptosis-related genes

Fig. 9

ROC curve of key lncRNA and mRNA: a NEAT1 in the discovery dataset (GSE198710);b NEAT1 in the validation set (GSE102541); c TLR4 in the discovery dataset (GSE58294) and d in the validation set (GSE16561)

Fig. 10

The relative expression of differentially expressed miRNA，mRNA, and lncRNA in SH-SY5Y cell: a Viability of SH-SY5Y cells after 6 h treatment with various concentrations of H2O2 (1 to 5000 μ M), estimated by CCK-8 assay; b miR-103a-3p; c miR-140-3p; d miR-17-5p; e TLR4; f NEAT1. The control group reflects the normal SH-SY5Y and the H2O2 group reflects the model, *P < 0.05, **P < 0.01, ***P < 0.001