Genome-wide profiling of miRNA-gene regulatory networks in mouse postnatal heart development—implications for cardiac regeneration

Figures

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  Figure 1. Experimental workflow. (A) Total RNA was collected on postnatal days 1, 4, 9, and 23 (P01, P04, P09 and P23, respectively) from mouse ventricles. RNA samples from 4 hearts/group were used in global miRNA expression profiling and data validation studies using qPCR. (B) Flow chart of data analysis and integration of miRNA sequencing data with transcriptomic data from (23) to identify target genes. Putative gene targets of miRNAs were verified from differentially regulated genes identified in our previous transcriptomic data analysis (23). DE, differentially expressed; FC, fold change.

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  Figure 2. Differential miRNA expression pattern during neonatal mouse heart development. Differentially expressed (fold change ≥1.8; FDR-adjusted p < 0.01) miRNAs are shown for time-point comparisons: P04–P01, P09–P04, P09–P01, and P23–P09. All miRNAs apart from miR-6236 remained up- or downregulated also in later time point comparisons, but are only listed for the first time point comparison in which differential expression was observed. *miR-6236 was downregulated in the P09-P01 comparison, but not in P23-P01 comparison.

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  Figure 3. Relative expression levels of differentially expressed miRNAs in postnatal mouse heart. (A) Expression levels of downregulated (fold change ≥1.8; FDR-adjusted p < 0.01) miRNAs in global miRNA profiling. (B) Expression levels of upregulated (fold change ≥1.8; FDR-adjusted p < 0.01) miRNAs in global miRNA profiling. The data are presented as normalized to P01 (mean ± STDEV; n = 4 animals). (C,D) validation of miRNA-sequencing results by qPCR for downregulated (C) and upregulated (D) miRNAs. Top 18 differentially expressed miRNAs from different time points were selected for validation. Cq values were normalized to the average value of two housekeeping miRNAs (miR-130a-3p and miR-28-5p) from the same sample. The data are shown as normalized to P01 (mean ± STDEV; n = 4 animals).

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  Figure 4. Comparison of miRNA expression in neonatal mouse cardiomyocytes, non-cardiomyocytes and ventricular tissue. The expression of miRNAs in mouse heart tissue and isolated cells was analyzed using qPCR and results are shown as relative miRNA expression for three comparisons: (A) cardiomyocytes (CM) vs. non-cardiomyocytes (non-CM); (B) ventricular tissue (VT) vs. CM; and (C) VT vs. non-CM. The data are presented as average ± standard error of the mean; n = 3. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

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  Figure 5. Predicted miRNA-gene regulatory networks in mouse hearts within the first 9 postnatal days. (A) Chord diagram showing that target genes of early downregulated miRNAs (P04–P01) were enriched in GO:BP and KEGG pathways (p ≤ 0.05) associated with cell cycle and proliferation. (B) The upregulated miR-150-5p showed negative regulatory effect on GO biological processes and KEGG pathways (p ≤ 0.05) related to cell cycle and proliferation in P01 to P09 time span. (C,D) Chord diagram demonstrating interaction network between enriched biological processes (C) and KEGG pathways (D) with downregulated miRNAs identified in P09–P04 and P09–P01 comparisons. The enrichment of the target genes of each miRNA with the GO biological processes and KEGG pathways (p ≤ 0.05) are presented with a single color. Gene-regulatory networks of miRNAs from P09–P01 comparison are shown in black color. The size of the section representing each GO term or KEGG pathway is proportional to the number of target genes associated with the corresponding GO term or KEGG pathway. (E) Sankey plot showing mitochondria-associated GO:CC enriched in verified target genes of downregulated miRNAs in the P09–P04 comparison.

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  Figure 6. Gene regulatory networks of downregulated miRNAs after postnatal day 9 and miRNAs associated with mitochondrial genes. (A,B) GO biological processes with enriched verified target genes of miRNAs that were downregulated in the P23–P09 comparison. The data has been divided into two chord diagrams for clarity of presentation. (C) KEGG pathways enriched by verified target genes of miRNAs downregulated in the P23–P09 comparison. (A–C) The enrichment of the target genes of each miRNA with the GO biological processes and KEGG pathways (p ≤ 0.05) are presented with a single color in the chord diagrams. The size of the section representing each GO term or KEGG pathway is proportional to the number of target genes associated with the corresponding GO term or KEGG pathway. (D) Chord diagram demonstrating that verified target genes of miRNAs enriched in mitochondria-associated GO cellular components. The enrichment of the target genes of each miRNA with the GO cellular component are presented with a single color. Gene-regulatory networks of miRNAs from P09–P01 comparison are shown in black color. (E) Sankey plot showing mitochondria-associated GO:CC enriched in verified target genes of…

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  Figure 7. Gene regulatory networks of upregulated miRNAs after postnatal day 9. Chord diagrams showing negative regulation of GO biological processes (A) and KEGG pathways (B) by target genes of upregulated miRNAs. The enrichment of the target genes of each miRNA with the GO biological processes and KEGG pathways (p ≤ 0.05) are presented with a single color in the chord diagrams. The size of the section representing each GO term or KEGG pathway is proportional to the number of target genes associated with the corresponding GO term or KEGG pathway.