A Network Comprised of miR-15b and miR-29a Is Involved in Vascular Endothelial Growth Factor Pathway Regulation in Thymus Adipose Tissue from Elderly Ischemic Cardiomyopathy Subjects

Figures

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  Figure 1. In silico analysis of miRNAs predicted to regulate VEGF-A. (a) MiRTargetLink Human was used to identify 44 miRNAs as strong potential VEGF-A regulators. (b) MiRTargetLink Human was used to identify 64 miRNAs as weak probability VEGF-A regulators. (c) MiRTargetLink Human was used to identify 87 miRNAs as probability VEGF-A regulators.

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  Figure 2. In silico analysis of angiogenic target genes. Target genes (green nodes) that could be regulated by miR-15b-5p; miR-16-5p; miR-29a-3p; miR-29b-3p; miR-195-5p; and miR-21-5p (fuchsia nodes) using MiRTargetLink Human to identify all potential mRNA target genes and the PANTHER database to identify those with a role in angiogenic processes. (a) Seventeen mRNAs were identified as potential miR-15b-5p target genes, of which two were angiogenesis related. (b) One hundred thirty-five mRNAs were identified as potential miR-16-5p target genes, of which nine were angiogenesis related. (c) Eighty-three mRNAs were identified as potential miR-29a-3p target genes, of which three were angiogenesis related. (d) Eighty mRNAs were identified as potential miR-29b-3p target genes, of which 10 were angiogenesis related. (e) Thirty-three mRNAs were identified as potential miR-195-5p target genes, of which three were angiogenesis related. (f) One hundred one mRNAs were identified as potential miR-21-5p target genes, of which five were angiogenesis related.

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  Figure 3. Expression profiles of miR-15b-5p, miR-16-5p, miR-29a-3p, miR-29b-3p, miR-195-5p, and miR-21-5p in human TAT and SAT. miRNA expression levels were measured by RT-qPCR, and the relative quantification of expression levels was carried out using the comparative threshold cycle (Ct) method. The hsa-RNU48 was used as an endogenous control. Data are expressed as the mean ± SEM and assessed using the Mann–Whitney U test to compare the elderly and middle-aged patient groups. (a) miRNAs were extracted from TAT (n = 18). (b) miRNAs were extracted from SAT (n = 18). * p ≤ 0.05.

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  Figure 4. Expression profiles of 18 potential angiogenic target genes of miR-15b-5p, miR-16-5p, miR-29a-3p, miR-29b-3p, miR-195-5p, and miR-21-5p. (a) mRNA expression level of BIRC5, GSK3B, JAG1, PDGFC, PIK3CG, RAF1, VEGFA and WNT7A). (b) mRNA expression level of FGF2, FGFR1, KDR, NOTCH2, PIK3R1 and STAT3. (c) mRNA expression level of FOS, PDGFRA, PDGFRB and RHOB. The mRNAs were extracted from TAT (n = 18). mRNAs expression levels were measured by RT-qPCR, and relative quantification of the expression levels was carried out with the comparative threshold cycle (Ct) method. EF1α was used as an endogenous control. Data are expressed as the mean ± SEM and assessed using the Mann–Whitney U test to compare the elderly and middle-aged patient groups. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

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  Figure 5. (a) Interaction network between miR-15b-5p, miR-16-5p, miR-29a-3p, miR-29b-3p, miR-195-5p, and miR-21-5p and their 12 angiogenic target genes in human TAT. Cytoscape v.3.2.1 software (NIGMS. 45 Center Drive, MSC 6200, Bethesda, Maryland 2089-6200) was used to draw the interaction network between miRNAs and their 12 angiogenic target genes differently expressed and considering the comparative results of expression levels of these miRNA/mRNAs between elderly and middle-aged patients. Grey nodes show the miRNAs that have similar expression levels in both elderly and middle-aged patients. Black nodes show the miRNAs that are upregulated in elderly patients compared with middle-aged patients; green nodes show the mRNAs that are downregulated in elderly patients compared with middle-aged patients; and red nodes show the mRNAs that are upregulated in elderly versus middle aged patients. Lines show potential interaction, direct or indirect, between miRNAs and their target genes; dots indicate potential upregulation of VEGFA; dashes indicate downregulated target genes; sine waves indicate potentially upregulated target genes. (b) Cytoscape was used to draw the putative interaction network…