Single-cell and bulk RNA sequencing-based screening and identification of extracellular trap network-related genes in neutrophils in acute myocardial infarction.

Background: The neutrophil-mediated generation of neutrophil extracellular traps (NETs) results in an augmented inflammatory response and cellular tissue injury during acute myocardial infarction (AMI). Through the analysis of public database information, we discovered and confirmed putative critical genes involved in NETs-mediated AMI.

Methods: The AMI dataset GSE66360 and the single-cell dataset GSE163465 were downloaded from the Gene Expression Omnibus database. Key genes were screened by bioinformatics. Quantitative real-time PCR (qRT-PCR) was used to verify the key genes, and then a Mendelian randomization (MR) study was conducted on the basis of the genome-wide association study to determine the causal relationship between key genes and AMI. Dimensionality reduction clustering, pseudo-time series, and cell communication were performed on the single-cell dataset to analyze the key genes screened by bulk RNA sequencing and the dynamic evolution of NETs in the AMI process. Immunohistochemistry and Western blot were used to verify the key genes.

Results: Six key genes, IL1β, S100A12, TLR2, CXCL1, CXCL2, and CCL4, were screened out through bioinformatics. qRT-PCR results showed that compared with the control group, the expression of 5 key genes was upregulated in the AMI group. In the MR study, CXCL1 and CCL4 were observed to have a causal relationship with AMI. Single-cell analysis showed that NETs-related genes CCL4, CXCL2, and IL1β were highly expressed. Combining single cells, qRT-PCR and MR, gene CCL4 was selected as the focus of the study. H9c2 cardiomyocytes simulated myocardial infarction under hypoxia, and the results showed that the expression of gene CCL4 was increased. The immunohistochemical results of gene CCL4 showed that the expression was upregulated in the AMI group.

Conclusions: We found 6 key genes related to NETs-mediated cell damage during AMI. The results of MR showed that CXCL1 and CCL4 were causally related to AMI. Combining single cells, qRT-PCR and MR, gene CCL4 may play an important role in the AMI process. Our results may provide some insights into neutrophil-mediated cell damage during AMI.