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. 2023 Jun 16:10:1082015.
doi: 10.3389/fcvm.2023.1082015. eCollection 2023.

PILRA is associated with immune cells infiltration in atrial fibrillation based on bioinformatics and experiment validation

Weihua Shi #  1 , Xiaoli Li #  1 , Yongxing Su  1 , Dezhao Liu  1 , Liying Wu  2 , Shuo Li  1 , Wenxiu He  1 , Guoqiang Zhong  1 , Zhiyuan Jiang  1
Affiliations

PILRA is associated with immune cells infiltration in atrial fibrillation based on bioinformatics and experiment validation

Weihua Shi et al. Front Cardiovasc Med. .

Abstract

Background and aims: inflammation plays an important role in atrial fibrillation (AF). In this study, we investigated the significance of immune cell infiltration in AF and identified the potential Hub genes involved in the regulation of immune cell infiltration in AF.

Methods: we obtained AF datasets from the GEO database and analyzed them for obtaining differentially expressed genes (DEGs) by R software. Then, we performed GO, KEGG, and GSEA enrichment analyses of DEGs. The Hub genes of AF were determined by least absolute shrinkage selection operator (LASSO) regression analysis and weighted gene co-expression network analysis (WGCNA). Their validation was verified by using quantitative polymerase chain reaction (qPCR) in the AF rat model. Finally, we used a single sample GSEA (ssGSEA) to analyze immune cell infiltration and its relationship with hub genes.

Results: We obtained 298 DGEs from the heatmap and found that DGEs were closely related to inflammation, immunity, and cytokine interactions by enrichment analyses. We obtained 10 co-expression modules by WGCNA. Among them, the module including CLEC4A, COTL1, EVI2B, FCER1G, GAPT, HCST, NCF2, PILRA, TLR8, and TYROBP had the highest correlation with AF. Four Hub genes (PILRA, NCF2, EVI2B, GAPT) were obtained further by LASSO analysis. The results suggested that the expression level of PILRA was significantly elevated in the rats with AF by qPCR, compared to the rats without AF. The results revealed that the infiltration of neutrophils, macrophages, monocytes, mast cells, immature B cells, myeloid-derived suppressor cell (MDSC), dendritic cell, and T cells and their partial subpopulations were closely related to AF by ssGSEA analysis, and PILRA was positively correlated with immature B cell, monocyte, macrophage, mast cell, dendritic cell, and T cells and their partial subpopulations by Spearman correlation analysis.

Conclusions: PILRA was closely related to multiple types of immune cell infiltration, which may be associated with AF. PILRA may be a novel target of intervention for AF.

Keywords: atrial fibrillation; immune cells 1nfiltration; inflammation; least absolute shrinkage selection operator (LASSO) regression analysis; weighted gene co-expression network analysis (WGCNA).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The study flowchart of data collection, analysis, processing, and experimental validation.
Figure 2
Figure 2
Identification differential expression genes of atrial fibrillation (A) heatmap of DGEs. (B) Volcano map of DGEs.
Figure 3
Figure 3
Enrichment analysis of DGEs in AF. (A) Gene ontology (GO) analysis of DEGs. (B) Kyoto encyclopedia of genes and genomes (KEGG) analysis of DEGs. (C) Gene set enrichment analysis (GSEA) of DEGs.
Figure 4
Figure 4
Construction of weighted gene co-expression network analysis (WGCNA) modules in AF. (A) The original and combined modules of tree diagram. (B) The black module has the highest correlation with AF in the heatmap of module-trait relationships. Red: positive correlations with AF; blue: negative correlations with AF. (C) The candidate genes contributing to AF in the black model shown in Scatter plot. When |GS| was more than 0.50 and |MM| was more than 0.80, the genes were chosen as the candidate genes. GS, gene significance; MM, module membership.
Figure 5
Figure 5
Least absolute shrinkage selection operator (LASSO) regression analysis of DGEs in AF. (A) Venn diagram of crossover genes between DEGs and candidate modules. (B) The trend of coefficient distribution of cross-validation. Four genes (PILRA, NCF2, EVI2B and GAPT) were obtained in the optimal λ value. (C) Distribution of pivotal genes in Lasso regression analysis.
Figure 6
Figure 6
Elevated expression levels of PILRA, EVI2B, NCF2, and GAPT in atrial tissue of patients with AF from GSE41177 and GSE79768. (A) The expression of PILRA in patients with AF and patients with sinus rhythm. (B) The expression of EVI2B in patients with AF and patients with sinus rhythm. (C) The expression of NCF2 in patients with AF and patients with sinus rhythm. (D) The expression of GAPT in patients with AF and patients with sinus rhythm. Con: the patients with sinus rhythm. AF: the patients with AF. (***p < 0.001; **p < 0.01; *p < 0.05).
Figure 7
Figure 7
Area under curve (AUC) of the Hub genes for diagnosis of AF. (A) AUC value of PILRA. (B) AUC value of GAPT. (C) AUC value of NCF2. (D) AUC value of EVI2B.
Figure 8
Figure 8
The electrocardiogram of rat with sinus rhythm (SR) or AF. (A) The representative electrocardiogram of rats with SR. p wave and regular R–R interval could be observed. (B) The representative electrocardiogram of rats with AF. p wave disappeared and was replaced by a disordered f wave with absolutely irregular R–R intervals. EB: transesophageal burst pacing.
Figure 9
Figure 9
Relative mRNA expression levels of Hub genes. (A) PILRA expression level in rats with or without AF. (B) EVI2B expression level in rats with or without AF. (C) GAPT expression level in rats with or without AF. (D) NCF2 expression level in rats with or without AF. (**p < 0.01; ns, no significant difference).
Figure 10
Figure 10
The association of PILRA with immune cell infiltration in AF. (A) Heatmap of immune cell infiltration in AF. (B)Violin plot showed the correlation between immune cells infiltration and AF. (C) The relationship between immune cells infiltration and PILRA.
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