Exploring novel biomarkers in dilated cardiomyopathy‑induced heart failure by integrated analysis and in vitro experiments

Figures

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  Figure 1. Flow chart of the study. In this study, two DCM-induced HF datasets were collected. WGCNA and differentially expressed genes analysis were used to identify key genes in the GSE79962 dataset. The relative fractions of infiltrated immune cells were evaluated the CIBERSORT algorithm. The relationship between key genes and immune cells was calculated by Spearman's correlation analysis. The potential function of the key module was explored using GO, GSEA and KEGG analysis. Moreover, key genes were validated in the GSE116250 dataset and in vitro experiments. The diagnostic efficacy of key genes was evaluated using a ROC curve in the GSE116250 dataset. DCM, dilated cardiomyopathy; GO, gene ontology; GSEA, Gene Set Enrichment Analysis; HF, heart failure; KEGG, Kyoto encyclopedia of genes and genomes; ROC, receiver operating characteristic; WGCNA, weighted gene co-expression network analysis.

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  Figure 2. Construction of gene co-expression modules. (A) Clustering dendrograms of 20 samples Red, 11 patients with dilated cardiomyopathy-induced heart failure; green, 9 healthy controls. (B) Finding appropriate soft-threshold powers (β). (C) Clustering of modules with similar expression profile. (D) Clustering dendrogram of genes by hierarchical clustering.

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  Figure 3. Key module selection and functional enrichment annotation. (A) Identification of the key modules associated with DCM-induced HF. Each row refers to a specific module. P-values and the corresponding correlation are stated in the cell. Blue represents negative correlation and red represents positive correlation. (B) Gene significance for DCM-induced HF in the blue module. (C) Kyoto Encyclopedia of Genes and Genomes pathway analysis of genes involved in the blue module. (D) GO functional term enrichment analysis of genes in the blue module. BP, biological process; CC, cellular component; DCM, dilated cardiomyopathy; HF, heart failure; GO, gene ontology; MF, molecular function.

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  Figure 4. Gene set enrichment analysis in the GSE79962 dataset from the Gene Expression Omnibus database. (A) AGE/RAGE signaling pathway in diabetic complications. (B) Th1 and Th2 cell differentiation. (C) Wnt signaling pathway. (D) ECM-receptor interaction. AGE, advanced glycation end product; ECM, extracellular matrix; RAGE, AGE receptor.

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  Figure 5. Identification of key genes. (A) Volcano plots of the significant DEGs between patients with DCM-induced HF and normal healthy control in the GEO dataset GSE79962. (B) Identification of common genes between hub genes identified in the WGCNA analysis and in the DEG analysis. (C) Comparison of expression levels of key genes between patients with DCM-induced HF and normal healthy controls in GSE79962. DCM, dilated cardiomyopathy; HF, heart failure; DEG, differentially expressed genes; FC, fold change; GEO, Gene Expression Omnibus; WGCNA, weighted gene co-expression network analysis.

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  Figure 6. Key gene validation in the Gene Expression Omnibus dataset GSE116250. mRNA expression levels of (A) SMOC2 and (B) PLCE1 were significantly increased in patients with DCM-induced HF compared with the control group. Expression levels of (C) SERPINA3, (D) MYH6, (E) S100A9, (F) LYVE1, (G) TUBA3D and (H) TUBA3E were significantly downregulated in patients with DCM-induced HF. DCM, dilated cardiomyopathy; HF, heart failure; LYVE1, lymphatic vessel endothelial hyaluronic acid receptor 1; MYH6, myosin heavy chain 6; PLCE1, phospholipase C ε1; S100A9, S100 calcium binding protein A9; SERPINA3, serpin family A member 3; SMOC2, cysteine-related modular calcium-binding protein 2; TUBA3, tubulin α3.

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  Figure 7. ROC curve analysis. The eight genes included (A) SMOC2, (B) PLCE1, (C) SERPINA3, (D) MYH6, (E) S100A9, (F) LYVE1, (G) TUBA3D, and (H) TUBA3E. AUC, area under the curve; LYVE1, lymphatic vessel endothelial hyaluronic acid receptor 1; MYH6, myosin heavy chain 6; PLCE1, phospholipase C ε1; ROC, receiver operating characteristic; S100A9, S100 calcium binding protein A9; SERPINA3, serpin family A member 3; SMOC2, cysteine-related modular calcium-binding protein 2; TUBA3, tubulin α3.

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  Figure 8. Extent of immune infiltration between patients with DCM-induced HF and those in the control group. (A) The relative proportion of various immune cells. (B) The heat map of different infiltrating immune cells. (C) The difference of infiltrating immune cells between patients with DCM-induced HF (red) and the control group (green). *P<0.05, **P<0.01 vs. non-failing donors. DCM-induced HF, dilated cardiomyopathy-induced heart failure; ns, not significant; ?, P-value cannot be calculated.

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  Figure 9. Correlations between key genes with immunocytes. Correlation between SMOC2 and (A) naive B cells and (B) monocytes. (C) Correlation between PLCE1 and naive B cells. (D) Correlation between MYH6 and monocytes. (E) Correlation between SERPINA3 and naive B cells. (F) Correlation between S100A9 and monocytes. (G) Correlation between LYVE1 and naive B cells. LYVE1, lymphatic vessel endothelial hyaluronic acid receptor 1; MYH6, myosin heavy chain 6; PLCE1, phospholipase C ε1; S100A9, S100 calcium binding protein A9; SERPINA3, serpin family A member 3; SMOC2, cysteine-related modular calcium-binding protein 2.

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  Figure 10. Construction of a cardiac injury model in vitro. (A) Cell viability following DOX treatment at various concentrations. ****P<0.0001 vs. 0 µM. (B) Protein expression levels of BCL2 and BAX protein in DOX-induced cardiac injury. **P<0.01 vs. control. (C) Apoptotic levels of AC16 cells in DOX-induced cardiac injury were determined by TUNEL assay. ***P<0.001 vs. control. Scale bar, 50 µm. (D) mRNA expression levels of ANP in DOX-induced cardiac injury. ***P<0.001 vs. control. (E) mRNA expression levels of BNP in DOX-induced cardiac injury. **P<0.01 vs. control. (F) Protein expression levels of ANP in DOX-induced cardiac injury model. **P<0.01 vs. control. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; DOX, doxorubicin.

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  Figure 11. Relative mRNA expression levels of key genes between control and DOX-induced cardiac injury cells. mRNA expression levels for (A) SMOC2, (B) PLCE1, (C) SERPINA3, (D) MYH6, (E) S100A9, (F) LYVE1, (G) TUBA3D, and (H) TUBA3E. **P<0.01, ***P<0.001, ****P<0.0001 vs. control. Control, untreated cells; DOX, doxorubicin; LYVE1, lymphatic vessel endothelial hyaluronic acid receptor 1; MYH6, myosin heavy chain 6; PLCE1, phospholipase C ε1; S100A9, S100 calcium binding protein A9; SERPINA3, serpin family A member 3; SMOC2, cysteine-related modular calcium-binding protein 2; TUBA3, tubulin α3.