Integration of miRNA and gene expression profiles suggest a role for miRNAs in the pathobiological processes of acute Trypanosoma cruzi infection

Figures

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  Figure 1. (a) Workflow of data processing and analysis used to identify DEMS-DEGs interactome: Mice were infected with 100 blood trypomastigotes of the Colombian strain of T. cruzi and the heart gene expression profiling was evaluated 15, 30 or 45 days after infection. The miRNA profile used in this study was from the same samples and previous published (Navarro et al. 2015). The numbers and letters in the workflow indicate each one of the analysis figures present in this paper. Gene expression, statistical analysis and identification of differentially expressed genes (DEG) was performed using Linear Model for microarray data (Limma) with adjustment for false discovery rate with the Benjamini-Hochberg method. (b) Principal component analysis (PCA) of gene expression was performed for all samples and all probe set, by using a median centering of the data set. The x-axis corresponds to principal component 1 (PC1) and the y-axis to the principal component 2 (PC2), the percentage of the variance is indicated between brackets. Based on their mRNA expression values, samples from each time group (non-infected, 15, 30 and 45 dpi) clustered together, thus confirming homogeneity of the gene…

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  Figure 2. Venn diagram showing the number of differentially expressed (a) genes and (b) enriched canonical pathways in each time point. The enriched canonical pathways were identified by Ingenuity Pathway (IPA) analysis or (c) gene set enrichment analysis software (GSEA). (c) Enrichment map for experimental Chagas disease: Pre-ranked lists were analyzed for enrichment in sets of functionally related genes (pathways). Enrichment map was drawn representing the enriched pathways (nodes) as networks where the color corresponds to z-score based activation state prediction blue:downregulated and red:upregulated). Node size represents the gene set size in that pathway.

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  Figure 3. Venn diagrams showing the number of differentially expressed mitochondrial genes, and/or genes belonging to the IFNγ- and Nrf2- modulated transcriptional profile. Genes in each transcriptional profile were obtained from published data and mitochondrial genes were obtained from Gene Ontology as described in the Methods section. Graphs display the number of differentially expressed genes at the three time points. The Venn diagram includes three colored circles. Blue circles indicate the number of DEGs that are IFNγ-modulated, green circles indicate the number of Nrf2-modulated/antioxidant response genes and red circles indicate the number of mitochondrial genes.

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  Figure 4. MiRNA target analysis and DEMs-DEGs Network. (a) Venn diagram showing the number of DEM targets (high predicted and experimentally validated targets) within the list of DEGs for each time point post infection as well as those that are shared among different time points. Red numbers indicate upregulated genes, blue numbers indicate downregulated genes. (b) A DEMs-DEGs network showing that individual targets associated with immunity, cardiovascular function or disease can be regulated by multiple miRNAs. The nodes are represented in graduation of red and green based on their fold change in expression at 30 dpi compared to the uninfected group.

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  Figure 5. Ingenuity Pathway Analysis (IPA) canonical pathways most significantly enriched in the heart of T. cruzi infected mice. The stacked bar chart displays the percentage of target DEGs molecules present in each pathway. The numerical value in the parentheses in front each pathway name represents the total number of genes in that canonical pathway. The Benjamini-Hochberg (B.-H.) method was used to adjust the right-tailed Fisher’s exact test P-value, which was always <0.001.

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  Figure 6. Upstream regulator analysis. Corrplot representation of the top10 upstream regulators. Cytokines and growth factors were only considered upstream regulators if were found to be differentially expressed and upregulated; otherwise they were excluded. Red or blue color represent activated or inhibited pathways, respectively, according to Z-score prediction a statistical calculation of the activation state.

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  Figure 7. (a) DEMs-DEGs networks related to the main Chagas disease clinicopathogical features. Networks related to myocarditis, fibrosis, arrhythmia and hypertrophy were built using IPA software. Each built network contains molecules represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Knowledge Base (IKB). The molecules are represented in graduation of red and green based on their fold change in expression at 30 dpi compared to 0 dpi. Each node shape represents one type of molecule, as follows: (b) Validation of miRNA target expression by qPCR. Genes chosen for validation were network nodes which were direct miRNA targets belonging to each of the pathophysiology networks. The genes validated were IFN-g (interferon gamma), VCAM1 (vascular cell adhesion molecule 1), CTSC (cathepsin C), IL-6 (interleukin 6), NOS2 (nitric oxide synthase 2), NTF3 (Neurotrophin 3), FASLG (FAS Ligand) and PLA2G4A (Phospholipase A2, Group IVA). The expression was calculated as the mean ± s.d. for each group as individual…