Bioinformatics and functional experiments reveal that MRC2 inhibits atrial fibrillation via the PPAR signaling pathway

Figures

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  Figure 1. Screening and functional enrichment analysis of GSE143924-DEGs. (A) Heatmap of 15 POAF samples in orange and 15 SR samples in blue. Each column represents sample data and each row a gene. (B,C) Sankey plot and dot plot of the BP and KEGG on up-regulated DEGs. (D,E) Sankey plot and dot plot of the BP and KEGG on down-regulated DEGs. POAF, postoperative atrial fibrillation after surgery; SR, sinus rhythm after surgery; GTPase, guanosine triphosphatase; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes; BP, biological process.

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  Figure 2. Identification of key module associated with AF in WGCNA. (A) Scale-free topology model (top) and average connectivity (bottom) to determine soft threshold power. (B) Gene clustering tree. (C) Sample clustering to detect outliers. (D) Cluster and eigengene adjacency heatmap for all modules. (E) Heatmap showing the relationship between different modules and clinical samples. AF, atrial fibrillation; WGCNA, weighted gene co-expression network analysis.

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  Figure 3. Identification of the overlapping genes from key module and GSE143924-DEGs. (A) Venn diagram of tan modules and DEGs to obtain the overlapping genes. (B-K) GSEA of 10 overlapping genes: (B) DSC2, (C) GAS7, (D) HTRA3, (E) LPCAT2, (F) LTBP1, (G) MRC2, (H) PAMR1, (I) PARD3, (J) SCARA5, (K) CDH19. DEGs, differentially expressed genes; ES, enrichment score; NP, normalized P value; GSEA, gene set enrichment analysis.

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  Figure 4. Expression level analysis on 10 overlapping genes. (A) MRC2. (B) DSC2. (C) GAS7. (D) CDH19. (E) LPCAT2. (F) SCARA5. (G) PARD3. (H) PAMR1. (I) HTRA3. (J) LTBP1. *, P<0.05; **, P<0.01. (K) ROC curve analysis on MRC2. AUC =0.800. TPR, true positive rate; FPR, false positive rate; AUC, area under the curve; CI, confidence interval; ROC, receiver operating characteristic.

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  Figure 5. Effect of over-MRC2 on the cell proliferation and apoptosis of AF. (A,B) The mRNA and protein expression levels of MRC2 in cells of HCFs induced by Ang II were lower than that of the control group. (C,D) The efficiency of MRC2 overexpression in cells of HCFs induced by Ang II was examined by qRT-PCR and WB assays. (E) The effect of over-MRC2 on cell proliferation was detected by CCK-8 in HCFs induced by Ang II. *, P<0.05; **, P<0.01. (F-I) The effect of overexpressed MRC2 on apoptosis was examined by flow cytometry in HCFs treated with Ang II. Q1 was necrotic cells, Q2 was late apoptotic cells, Q3 was early apoptotic cells, and Q4 was live cells. Ang II, angiotensin II; NC, negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; over-, over-expressed-; OD, optical density; FITC, fluorescein isothiocyanate; PI, propidium iodide; mRNA, messenger RNA; HCFs, human cardiac fibroblasts; qRT-PCR, quantitative real-time polymerase chain reaction; WB, western blotting; CCK-8, cell counting kit-8.

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  Figure 6. Knockdown of MRC2 promoted the proliferation of Ang II-induced HCFs. (A) The level of MRC2 was knocked down in HCFs. (B) MRC2 knockdown in Ang II-induced HCFs. (C) The effects of Ang II treatment and si-MRC2-1 transfection on the proliferation of HCFs were detected by CCK-8. *, P<0.05; **, P<0.01; #, P<0.05, compared between Ang II + si-NC and Ang II + si-MRC2-1 groups. (D-G) The protein expression levels of (D) PPARα, (E) PPARγ, (F) CPT-1, and (G) SIRT3 in Ang II-induced HCFs treated with over-MRC2. *, P<0.05; **, P<0.01; ***, P<0.001; #, P<0.05, compared with Ang II group. (H) The effect of over-MRC2 on the protein expression levels of PPARα, PPARγ, CPT-1, and SIRT3 in Ang II-induced HCFs as detected by WB method. Si-, small interfering-; NC, negative control; Ang II, angiotensin II; OD, optical density; over-, over-expressed-; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCFs, human cardiac fibroblasts; CCK-8, cell counting kit-8; WB, Western blotting.