Table 1.
Primer sequences for qRT-PCR assay.
Fig 1.
Screening of DEGs and analysis of co-expression network in GSE42148 gene expression profile.
(A) Volcano plot of the 2044 DEGs identified from the GSE42148 gene expression profile, showing 1057 up-regulated and 987 down-regulated DEGs. (B) Analysis of the scale-free fit index for various soft-thresholding powers (β), with β = 28 selected as the most optimal value ensuring a scale-free network. (C) Cluster dendrogram of the DEGs, depicting a total of 8 color-coded modules (excluding the grey module), each representing a cluster of highly interconnected genes. (D) Eigengene adjacency heatmap illustrating the relationships among the identified gene modules. (E) Heatmap of the correlation between the gene modules and the two sample groups in the GSE42148 dataset, orange indicates a positive correlation and blue indicates a negative correlation.
Fig 2.
Enrichment analysis and PPI network analysis for key modules.
(A-B) Bubble plots, Wikipathway enrichment analysis of genes in (A) the black module and (B) the turquoise module, the size of each bubble represents the gene count and the color represents the adjusted p-value. (C-E) Visualization of the PPI network of the top 30 genes identified by the BottleNeck (C), Degree (D), and EcCentricity (E) algorithms. In each network, nodes represent genes, edges represent protein-protein interactions, and the node size reflects the connection degree within the network.
Fig 3.
Identification and validation of overlapping genes with potential clinical diagnostic value for CHD.
(A) Venn diagram of six overlapping genes, namely IMP3, IFNG, CASP3, RNASEH2A, UMPS, and BATF, identified from the PPI networks generated by the three algorithms. (B) Bar graph, significant downregulation of these six genes in case groups compared to controls in the GSE42148 dataset. *p<0.05, **p<0.01, ***p<0.001. (C-H) ROC curve analysis of six genes (C) IFNG, (D) BATF, (E) CASP3, (F) IMP3, (G) RNASEH2A, and (H)UMPS, the horizontal axis is 1-Specificities, and the vertical axis is Sensitivities. The AUC value under the ROC curve is used to evaluate the performance of the model, and the closer to 1, the better the model performance.
Fig 4.
BATF alleviates the damage caused by ox-LDL induction in HCAEC.
(A) qRT-PCR analysis of BATF mRNA expression in HCAEC cells following ox-LDL treatment. (B) WB analysis of BATF protein levels in HCAEC cells after ox-LDL treatment. (C) Efficiency of BATF overexpression in HCAEC cells post ox-LDL treatment, as assessed by qRT-PCR. (D) Efficiency of BATF knockdown in HCAEC cells post ox-LDL treatment, as determined by qRT-PCR. (E) Flow cytometry analysis of apoptosis in HCAEC cells post ox-LDL treatment, with either BATF overexpression or knockdown. Quadrants represent different stages of cell death: Lower left, live cells; lower right, early apoptotic cells; upper right, late apoptotic cells; upper left, necrotic cells. (F) WB analysis of apoptosis-related proteins Bcl-2 and Bax in HCAEC cells after ox-LDL treatment and BATF overexpression or knockdown. (G) LDH activity in homogenates of HCAEC cells post ox-LDL treatment and either BATF overexpression or knockdown, as measured by the LDH assay kit. *p<0.05.
Fig 5.
BATF transcriptionally activates SIRT1 expression in ox-LDL-treated HCAEC.
(A) qRT-PCR analysis of SIRT1 mRNA expression level in HCAEC after ox-LDL treatment. (B) WB analysis detected the expression level of SIRT1 protein in HCAEC after ox-LDL treatment. (C) The correlation map of the JASPAR database shows the DNA-binding motif of BATF and the binding site with SIRT1. (D) Dual-luciferase reporter assay demonstrates luciferase activity in cells co-transfected with BATF overexpression and SIRT1 3’UTR WT. (E) Dual luciferase reporter assays luciferase activity in cells co-transfected with si-BATF#1 and SIRT1 3’UTR WT. (F) ChIP assay detected DNA affinity for BATF and SIRT1 promoter regions. *p<0.05, **p<0.01.
Fig 6.
Interaction between BATF and SIRT1 regulates ox-LDL-induced apoptosis and cell injury in HCAEC.
(A) The apoptosis of HCAEC with OE-BATF+SIRT1 inhibitor was detected by flow cytometry after ox-LDL treatment. (B) Flow cytometry analysis detected apoptosis of HCAECs treated with si-BATF#1+SIRT1 activator after ox-LDL treatment. (C) LDH kit detects LDH activity of homogenized cells in HCAEC with OE-BATF+SIRT1 inhibitor after ox-LDL treatment. (D) The LDH kit detects the LDH activity of homogenized cells in HCAEC with si-BATF#1+SIRT1 activator after ox-LDL treatment.