TRIM13 prevents cardiomyocyte injury by affecting apoptosis through interacting with CD3D in myocardial infarction

Figures

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  Figure 1. Bioinformatics analysis flowchart

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  Figure 2. The results of differential gene analysis (A) Volcanic map of differential genes (DEG1). (B) Heatmap of differential genes (DEG1). (C) Volcanic map of differentially lncRNAs. (D) Heatmap of differentially lncRNAs. (E) Volcanic map of differentially circRNAs. (F) Heatmap of differentially circRNAs. (G) Volcanic map of differential genes (DEG2). (H) Heatmap of differential genes (DEG2).

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  Figure 3. Function enrichment analysis of differentially expressed genes related to MI (A) Venn diagram between DEG1 and DEG2. (B) GO function analysis of genes. (C) TreeMap of GO function analysis of genes. (D) Bubble chart of KEGG function analysis of genes. (E) TreeMap of KEGG function analysis of genes. (F) PPI network. (G) Degree algorithm (Top 10). (H) MCC algorithm (Top 10). (I) MNC algorithm (Top 10). (J) Venn of 3 algorithms.

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  Figure 4. Expression validation of key genes (A) The expression of key genes in chip data of heart tissue samples. (B) The expression of key genes in GSE236374. (C) Detected the expression of key genes in the mouse heart tissue by RT-qPCR. vs. control group or Sham group, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (n = 6). Data are represented as mean ± SD.

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  Figure 5. GeneMANIA and disease association analysis (A) GeneMANIA analysis. (B) Functional similarity analysis of key genes, the x axis represented the similarity index, default threshold = 0.75, and the y axis represented key genes. (C) Association analysis between key genes with cardiovascular-related diseases. (D) Association analysis between key genes with MI.

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  Figure 6. Gene Set Enrichment Analysis (GSEA) (A) GSEA of Cd8b1. (B) GSEA of Pdcd1. (C) GSEA of Klrd1. (D) GSEA of Klrk1. (E) GSEA of Cd3g. (F) GSEA of Cd74. (G) GSEA of Cd3d. Different color curves represented different KEGG pathways, the Y axis represented a degree of enrichment, and the vertical line represented the genes enriched in the pathway.

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  Figure 7. Regulation and interaction network of key genes (A) Information network of key genes. (B) Sankey of TF-mRNA, left were TFs, right were mRNAs. (C) Network of mRNA-TF, blue represented key genes, and yellow represented TFs. (D) Network of mRNA-kinases, dark blue represented mRNAs, while light blue represented kinases. (E) Network of TFs-kinases, yellow represented TFs, while light blue represented kinases. (F) TF-kinases-mRNA, red represented kinases that commonly regulate mRNA and TF. (G) Network of lncRNA-miRNA-mRNA. (H) Network of circRNA-miRNA-mRNA. Blue represented mRNAs, green represented miRNAs, purple represented lncRNAs, and yellow represented circRNAs. (I) Small molecule drug predicted, blue represented genes, yellow represented drugs, red represented diseases.

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  Figure 8. Knockdown of CD3D could relieve OGD-induced cardiomyocyte apoptosis (A and B) Detection of transfection efficiency by RT-qPCR and western blot. (C) CCK-8 assay. (D) Detected the levels of LDH and cTnT by ELISA. (E) Detected the levels of IL-1β, IL-6, and TNF-α by ELISA. (F) Detected the expression of cTnT by IF (Scale bars, 20 μm). (G) Detection of cell apoptosis rate by flow cytometry. (H) Detected the expression of cleaved-caspase-3, Bax and Bcl-2 by western blot. ∗∗p < 0.01, ∗∗∗p < 0.001, vs. NC group. #p < 0.05, ##p < 0.01, ###p < 0.001, vs. OGD group (n = 3). Data are represented as mean ± SD.

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  Figure 9. TRIM13 interacted with CD3D and regulated its ubiquitination (A) CO-IP. (B) GST pull-down. (C and D) Detection of transfection efficiency by RT-qPCR and western blot. (E) Detected the expression of TRIM13 and CD3D by western blot. (F) Ubiquitin level analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (n = 3). Data are represented as mean ± SD.

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  Figure 10. Knockdown of TRIM13 relieved OGD-induced cardiomyocyte apoptosis through downregulation of CD3D (A and B) Detection of transfection efficiency by RT-qPCR and western blot. (C and D) Detected the expression of CD3D by RT-qPCR and western blot. (E) CCK-8 assay. (F) Detected the levels of LDH and cTnT by ELISA. (G) Detected the levels of IL-1β, IL-6, and TNF-αby ELISA. (H) Detected the expression of cTnT by IF (Scale bars, 20 μm). (I) Detection of cell apoptosis rate by flow cytometry. (J) Detected the expression of cleaved-caspase-3, Bax and Bcl-2 by western blot. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, vs. OGD group. #p < 0.05, ##p < 0.01, ###p < 0.001, vs. si-TRIM13 group (n = 3). Data are represented as mean ± SD.

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  Figure 11. Knockdown of CD3D could alleviate the progression of MI mice (A) Left ventricular FS and EF of the MI mouse. (B) HE staining (Scale bars, 50 μm). (C) TTC staining. (D) Detected the expression of TRIM13 and CD3D by western blot. (E) Detected the levels of LDH and cTnT by ELISA. (F) Detected the levels of IL-1β, IL-6, and TNF-αby ELISA. (G) Detected the expression of cTnT by IF (Scale bars, 20 μm). (H) TUNEL staining (Scale bars, 20 μm). (I) Detected the expression of cleaved-caspase-3, Bax and Bcl-2 by western blot. ∗∗p < 0.01, ∗∗∗p < 0.001, vs. Sham group. #p < 0.05, ##p < 0.01, vs. MI group (n = 6). Data are represented as mean ± SD.