The Effect of N6-Methyladenosine Regulators and m6A Reader YTHDC1-Mediated N6-Methyladenosine Modification Is Involved in Oxidative Stress in Human Aortic Dissection

Abstract

Aortic dissection (AD) develops pathological changes in the separation of the true and false aortic lumen, with high lethality. m6A methylation and oxidative stress have also been shown to be involved in the onset of AD. Through bioinformatics methods, three differentially expressed m6A regulators (YTHDC1, YTHDC2, and RBM15) were excavated from the GSE52093 dataset in the Gene Expression Omnibus (GEO) database, and functional enrichment analysis of the differentially expressed genes (DEGs) regulated by m6A regulators was performed. Then, the genes with oxidative stress-related functions among these genes were found. The protein interaction network of the oxidative stress-related genes and the competing endogenous RNA- (ceRNA-) miRNA-mRNA network were constructed. Among them, DHCR24, P4HB, and PDGFRA, which have m6A differences in AD samples, were selected as key genes. We also performed immune infiltration analysis, as well as cell-gene correlation analysis, on samples from the dataset. The results showed that YTHDC1 was positively correlated with macrophage M1 and negatively correlated with macrophage M2. Finally, we extracted AD and healthy aorta RNA and protein from human tissues that were taken from AD patients and patients who received heart transplants, performed quantitative real-time PCR (qRT-PCR) on YTHDC2 and RBM15, and performed qRT-PCR and western blot (WB) detection on YTHDC1 to verify their differences in AD. The mRNA and protein levels of YTHDC1 were consistent with the results of bioinformatics analysis and were downregulated in AD. Immunofluorescence (IF) was used to colocalize YTHDC1 and endothelial cell marker CD31. After knocking down YTHDC1 in human umbilical vein endothelial cells (HUVECs), reactive oxygen species (ROS) levels had a tendency to increase and the expression of peroxide dismutase SOD2 was decreased. This study provides assistance in discovering the role of m6A regulator YTHDC1 in AD. In particular, m6A modification participates in oxidative stress and jointly affects AD.

Figure 1

Study workflow.

Figure 2

Identification of DEMRGs and DEGs. (a) Volcano plot of DEGs in GSE52093. (b) Heat map of differential expression of m6A regulators. (c) Expression boxplot of m6A regulators. “^∗” means adj. P value < 0.05.

Figure 3

GO function and KEGG pathway enrichment results of DEGs regulated by DEMRGs.

Figure 4

The network of oxidative stress-related genes regulated by DEMRGs. (a) Network of the relationship between DEMRGs and oxidative stress-related genes. (b) PPI network of oxidative stress-related genes: genes in yellow were upregulated, and genes in blue were downregulated.

Figure 5

The ceRNA network of mRNA-miRNA-lncRNA for DEMRGs: yellow stands for DEMRGs, purple stands for miRNAs, and green stands for ceRNAs (lncRNAs).

Figure 6

Identification of DEMRGs. (a) Coefficient plot of LASSO regression. (b) Cross verification curve of LASSO regression. (c) ROC curve analysis of DEMRGs.

Figure 7

Identification of key m6A oxidative stress-related genes. (a) Boxplot of expression of key m6A oxidative stress-related genes in GSE153434: “^∗” means P < 0.05, “^∗∗∗” means P < 0.001, and “^∗∗∗∗” means P < 0.0001. (b) Nomo-diagram-logistic model. (c) Calibration curve analysis. (d) Decision curve analysis. (e) Clinical impact analysis.

Figure 8

Interaction of drugs with P4HB and PDGFRA: the size of the shape represents the degree of interaction.

Figure 9

Correlations between genes and immune cells. (a) Correlation between DEMRGs and immune cells. (b) The correlation between key genes and immune cells. (c) The regulatory relationship between DEMRGs and key genes. (d) Correlation between DEMRGs and key genes.

Figure 10

Human aortic tissue validation, qRT-PCR results of DEMRGs: “^∗” means P < 0.05.

Figure 11

Human aortic tissue validation, WB results of YTHDC1. (a) Western blot in AD and normal. (b) Significance test of difference: “^∗” means P < 0.05.

Figure 12

IF staining of DAPI, YTHDC1, and CD31 and their merged images.

Figure 13

Effects of YTHDC1 on ROS and inflammation. “^∗” means P < 0.05, “^∗∗” means P < 0.01, and “^∗∗∗∗” means P < 0.0001. (a) YTHDC1 knockdown efficiency. (b) ROS levels of the si-YThDC1 groups and si-NC groups. (c) SOD2 relative expression of the si-YTHDC1 groups and si-NC groups. (d) PRR (NOD1, NOD2, and NLRP3) relative expression of the si-YTHDC1 groups and si-NC groups.