Identification of a novel immune infiltration-related gene signature, MCEMP1, for coronary artery disease

Abstract

Background: This study aims to identify a novel gene signature for coronary artery disease (CAD), explore the role of immune cell infiltration in CAD pathogenesis, and assess the cell function of mast cell-expressed membrane protein 1 (MCEMP1) in human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (ox-LDL).

Methods: To identify differentially expressed genes (DEGs) of CAD, datasets GSE24519 and GSE61145 were downloaded from the Gene Expression Omnibus (GEO) database using the R "limma" package with p < 0.05 and |log2 FC| > 1. Gene ontology (GO) and pathway analyses were conducted to determine the biological functions of DEGs. Hub genes were identified using support vector machine-recursive feature elimination (SVM-RFE) and least absolute shrinkage and selection operator (LASSO). The expression levels of these hub genes in CAD were validated using the GSE113079 dataset. CIBERSORT program was used to quantify the proportion of immune cell infiltration. Western blot assay and qRT-PCR were used to detect the expression of hub genes in ox-LDL-treated HUVECs to validate the bioinformatics results. Knockdown interference sequences for MCEMP1 were synthesized, and cell proliferation and apoptosis were examined using a CCK8 kit and Muse^® Cell Analyzer, respectively. The concentrations of IL-1β, IL-6, and TNF-α were measured with respective enzyme-linked immunosorbent assay (ELISA) kits.

Results: A total of 73 DEGs (four down-regulated genes and 69 up-regulated genes) were identified in the metadata (GSE24519 and GSE61145) cohort. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results indicated that these DEGs might be associated with the regulation of platelet aggregation, defense response or response to bacterium, NF-kappa B signaling pathway, and lipid and atherosclerosis. Using SVM-RFE and LASSO, seven hub genes were obtained from the metadata. The upregulated expression of DIRC2 and MCEMP1 in CAD was confirmed in the GSE113079 dataset and in ox-LDL-treated HUVECs. The associations between the two hub genes (DIRC2 and MCEMP1) and the 22 types of immune cell infiltrates in CAD were found. MCEMP1 knockdown accelerated cell proliferation and suppressed cell apoptosis for ox-LDL-treated HUVECs. Additionally, MCEMP1 knockdown appeared to decrease the expression of inflammatory factors IL-1β, IL-6, and TNF-α.

Conclusions: The results of this study indicate that MCEMP1 may play an important role in CAD pathophysiology.

Keywords: Coronary artery disease; Immune cell infiltration; MCEMP1; Machine learning algorithm; ox-LDL treatment HUVECs cells.

Figure 1. Screening and enrichment analyses of DEGs in CAD.

(A) Volcano plot of DEGs in CAD. The x-axis represents log2FC, and the y-axis represents -log10 (p-value). Green dots represent downregulated genes, and red dots represent upregulated genes. (B) Heatmap of DEGs. The x-axis represents the samples, and the y-axis represents DEGs. Red indicates upregulated, and blue represents downregulated. The darker the color, the higher the expression level. (C) Bar chart for GO enrichment analysis, including biological process, cellular component, and molecular function (top 10, respectively). (D) Bubble diagram for KEGG enrichment of DEGs (top 10). DEGs, differentially expressed genes; CAD, coronary artery disease; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 2. Selection and verification of diagnostic hub genes.

(A) Accuracy of candidate gene selection in the SVM-RFE algorithm. (B) Coefficient profiles of candidate genes in the LASSO model. (C) Venn diagram showing the overlap of characteristic genes of the two algorithms. (D) Bar chart for validation of the expression of candidate diagnostic markers in the GSE113079 dataset. Red represents the case group and blue represents the control group. *p < 0.05; ***p < 0.001; NS, not significant. SVM-RFE, support vector machine-recursive feature elimination; LASSO, least absolute shrinkage and selection operator.

Figure 3. Correlation of two diagnostic markers with immune cell infiltration and signal pathway.

Lollipop chart for immune cell infiltration with DIRC2 (A) and MCEMP1 (B). Line graph GSEA analysis for the top five pathways of DIRC2 (C) and MCEMP1 (D).

Figure 4. Establishment of ox-LDL-treated HUVECs.

(A) Cellular viability was detected by CKK-8 assay, which was normalized to the control group. (B and C) LDH and NO levels in ox-LDL-treated HUVECs were analyzed by chromometry using commercially available assay kits. (D) The expression of ICAM-1 was detected by western blot assay. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. The expression of DIRC2 and MCEMP1 in ox-LDL-treated HUVECs.

(A) The mRNA and protein expression of DIRC2 and MCEMP1 in ox-LDL-treated HUVECs by qRT‐PCR assay and Western blot assay. *p < 0.05; **p < 0.01; ***p < 0.001. ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; qRT‐PCR, quantitative real‐time polymerase chain reaction.

Figure 6. Low expression of MCEMP1 promoted the dysfunction of ox-LDL-treated HUVECs.

The expression of MCEMP1 in ox-LDL-treated HUVECs by qRT-PCR (A) and Western blot (B). The effect of MCEMP1 knockdown on cell viability by CCK-8 assay. (C) The effect of MCEMP1 knockdown on cell apoptosis by Muse^® Cell Analyzer. (D) The levels of IL-1β, IL-6, and TNF-α in ox-LDL-treated HUVECs by ELISA Kits. *p < 0.05; **p < 0.01; ***p < 0.001. ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; qRT‐PCR, quantitative real‐time polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.