Identification of core cuprotosis-correlated biomarkers in abdominal aortic aneurysm immune microenvironment based on bioinformatics

Abstract

Background: The occurrence of abdominal aortic aneurysms (AAAs) is related to the disorder of immune microenvironment. Cuprotosis was reported to influence the immune microenvironment. The objective of this study is to identify cuprotosis-related genes involved in the pathogenesis and progression of AAA.

Methods: Differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) in mouse were identified following AAA through high-throughput RNA sequencing. The enrichment analyses of pathway were selected through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). The validation of cuprotosis-related genes was conducted through immunofluorescence and western blot analyses.

Results: Totally, 27616 lncRNAs and 2189 mRNAs were observed to be differentially expressed (|Fold Change| ≥ 2 and q< 0.05) after AAA, including 10424 up-regulated and 17192 down-regulated lncRNAs, 1904 up-regulated and 285 down-regulated mRNAs. Gene ontology and KEGG pathway analysis showed that the DElncRNAs and DEmRNAs were implicated in many different biological processes and pathways. Furthermore, Cuprotosis-related genes (NLRP3, FDX1) were upregulated in the AAA samples compared with the normal one.

Conclusion: Cuprotosis-related genes (NLRP3,FDX1) involved in AAA immune environment might be critical for providing new insight into identification of potential targets for AAA therapy.

Keywords: Abdominal aortic aneurysm; Cuprotosis; FDX1; NLRP3; immune environment.

Figure 1

The workflow of this study.

Figure 2

Expression profiles of LncRNAs and mRNAs in mouse after AAA. Scatter-plot for comparing global expression profiles of lncRNAs (A) and mRNAs (C) in the artery between the AAA and sham-operated mice. X and Y axes indicate the mean normalized signal values (log2 scaled). Blue points represent up-regulated lncRNAs or mRNAs while red points represent downregulated lncRNAs or mRNAs. The heat map shows hierarchical clustering of DElncRNAs (B) and DEmRNAs (D) in the artery between the AAA and sham-operated mice. The color scale indicates the expression of DElncRNAs and DEmRNAs.

Figure 3

Circos plots representing the distribution of DElncRNAs and DEmRNAs on mice chromosomes. The outermost layer of the circos plot is the chromosome map of the rat genome. The largest and larger inner circles represent all DElncRNAs detected by RNA-sequencing with fold change ≥2.0, p < 0:05, and FDR < 0.05. The increased or decreased lncRNAs are marked with red or green bars, respectively, and bar heights in the larger inner circle indicate numbers of DElncRNAs. The smaller and smallest inner circles represent all DEmRNAs detected by RNA-sequencing with fold change ≥2.0, p < 0:05 and FDR < 0.05. Increased or decreased mRNAs are marked with red or green bars, respectively, and bar heights in the smallest inner circle indicate numbers of DEmRNAs.

Figure 4

qRT-PCR validation of DElncRNAs and DEmRNAs in the AAA mice compared with matched tissues of sham-operated mice. **p value < 0.01, ***p value < 0.001

Figure 5

GO enrichment analysis for the DEmRNAs with the 10 highest enrichment scores. (A) GO enrichment analysis for co-expressed DEmRNAs of DElncRNAs. Blue bars are biological processes, green bars are cellular components, and yellow bars are molecular functions. (B)The size of the spot indicates the gene numbers enriched in the pathway, and the color of the spot indicates the significance level of the enriched pathway.

Figure 6

Functional annotation and KEGG pathway enrichment analyses for co-expressed DEmRNAs of DElncRNAs. The size of the spot indicates the gene numbers enriched in the pathway, and the color of the spot indicates the significance level of the enriched pathway. The abscissa is the enrichment score. Size represents the number of enriched genes, and color indicates the degree of enrichment. Higher enrichment scores correlate with lower P-values, indicating that the enrichment of differentially expressed genes in given pathway is significant.

Figure 7

Validation of the expression of FDX1 and NLRP3 in Ang II induced AAA model. (A) The immunofluorescence images of FDX1 and NLRP3 on abdominal aorta in Sham group and AAA group. Scale bar, 50 μm and 100 μm. L: Tunica Lumen; M: Tunica Medium; A: Tunica Adventitia. (B) Quantitative analysis of FDX1 fluorescence intensity in the two groups. (C) Quantitative analysis of NLRP3 fluorescence intensity in the two groups. (D–F) Western blot analysis of FDX1 and NLRP3 on abdominal aorta in Sham group and AAA group. (G–I) Western blot analysis of FDX1 and NLRP3 on VSMCs in Control group and Ang II group. Repetition=3, **p value < 0.01, ***p value < 0.001.