Identification of Cardiac CircRNAs in Mice With CVB3-Induced Myocarditis

Figures

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  FIGURE 1. Identification of differentially expressed circRNAs in viral FM and NFM. (A) The flowchart of the present study. (B) Cardiac leukocyte infiltration and myocyte necrosis were detected by H&E staining in A/J or C57BL/6 mice respectively. (C) Inflammation score and necrosis area were performed for evaluating the severity of myocarditis; *p < 0.05. (D) Hierarchical cluster analysis of circRNAs between CVB3 and control groups. Red strip, high relative expression; green strip, low relative expression; Color brightness reflects the degree of expression increase or decrease. (E) Volcano plot of the expression of circRNAs in heart tissues. The red or blue plots represent up- or down-regulated circRNAs with p < 0.05, whereas the black plots represent insignificant changes. (F) Venn diagrams showed overlap of abnormally expressed circRNAs between A/J and C57BL/6 myocarditis models.

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  FIGURE 2. Validation of differentially expressed circRNAs in FM and NFM. (A,B) A/J-specific upregulated (A) and downregulated circRNAs (B) were verified by qRT-PCR in another heart samples; *p < 0.05 vs. Control. (C,D) C57BL/6-specific upregulated (C) and downregulated (D) circRNAs were verified by qRT-PCR in another heart samples; *p < 0.05 vs. Control. (E,F) The tissue distribution of A/J-specific (E) and C57BL/6-specific (F) dysregulated circRNAs; *p < 0.05 vs. Heart.

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  FIGURE 3. Correlation between circRNAs expression levels and cardiac inflammation. (A) The correlation between A/J-specific upregulated circRNAs (mmucirc_009765, mmucirc_015278, mmucirc_010217) and inflammatory response, N > 12. (B) The correlation between A/J-specific downregulated circRNAs (mmucirc_006378, mmucirc_016699, mmucirc_016687) and inflammatory response, N > 12. (C) The correlation between C57BL/6-specific upregulated circRNAs (mmucirc_020019, mmucirc_000569, mmucirc_001485) and inflammatory response, N > 12. (D) The correlation between C57BL/6-specific downregulated circRNAs (mmucirc_018655, mmucirc_016919, mmucirc_018173) and inflammatory response, N > 12.

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  FIGURE 4. Correlation between circRNAs expression levels and cardiac function. (A) The correlation between A/J-specific upregulated circRNAs (mmucirc_009765, mmucirc_015278, mmucirc_010217) and cardiac function, N > 12. (B) The correlation between A/J-specific downregulated circRNAs (mmucirc_006378, mmucirc_016699, mmucirc_016687) and cardiac function, N > 12. (C) The correlation between C57BL/6-specific upregulated circRNAs (mmucirc_020019, mmucirc_000569, mmucirc_001485) and cardiac function, N > 12. (D) The correlation between C57BL/6-specific downregulated circRNAs (mmucirc_018655, mmucirc_016919, mmucirc_018173) and cardiac function, N > 12.

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  FIGURE 5. Bioinformatic analysis of circRNAs in viral myocarditis. (A,B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for cis-target genes of A/J-specific (A) and C57BL/6-specific (B) dysregulated circRNAs. (C) CircRNA-miRNAs networks for A/J-specific dysregulated circRNAs. (D) KEGG pathway analyses for miRNAs targeted by A/J-specific dysregulated circRNAs. (E) CircRNA-miRNAs networks for C57BL/6-specific dysregulated circRNAs. (F) KEGG pathway analyses for miRNAs targeted by C57BL/6-specific dysregulated circRNAs.

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  FIGURE 6. Silencing of circArhgap32 promotes cardiomyocytes apoptosis. (A) Schematic illustrating the structure of circSnx5. (B) CircArhgap32 transcript was validated by Sanger sequencing. (C) Total RNA was digested with or without RNase R, followed by qRT-PCR measurements of circArhgap32, Arhgap32 and GAPDH; *p < 0.05 vs. RNase R (−). (D) Cell viability was detected in si-Arhgap32 or siRNA-NC treated HL-1 cells; *p < 0.05 vs. siRNA-NC. (E) Western blot for Bax and Bcl2 expression in si-Arhgap32 or siRNA-NC treated HL-1 cells; *p < 0.05 vs. siRNA-NC. (F) Cell apoptosis was detected by flow cytometry; *p < 0.05 vs. siRNA-NC.