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. 2016 Dec 16;6:37446.
doi: 10.1038/srep37446.

Time Series miRNA-mRNA Integrated Analysis Reveals Critical miRNAs and Targets in Macrophage Polarization

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Free PMC article

Time Series miRNA-mRNA Integrated Analysis Reveals Critical miRNAs and Targets in Macrophage Polarization

Liangqun Lu et al. Sci Rep. .
Free PMC article

Abstract

Polarization of macrophages is regulated through complex signaling networks. Correlating miRNA and mRNA expression over time after macrophage polarization has not yet been investigated. We used paired RNA-Seq and miRNA-Seq experiments to measure the mRNA and miRNA expression in bone marrow-derived macrophages over a time-series of 8 hours. Bioinformatics analysis identified 31 differentially expressed miRNAs between M1 and M2 polarized macrophages. The top 4 M1 miRNAs (miR-155-3p, miR-155-5p, miR-147-3p and miR-9-5p) and top 4 M2 miRNAs (miR-27a-5p, let-7c-1-3p, miR-23a-5p and miR-23b-5p) were validated by qPCR. Interestingly, M1 specific miRNAs could be categorized to early- and late-response groups, in which three new miRNAs miR-1931, miR-3473e and miR-5128 were validated as early-response miRNAs. M1 polarization led to the enrichment of genes involved in immune responses and signal transduction, whereas M2 polarization enriched genes involved in cell cycle and metabolic processes. C2H2 zinc-finger family members are key targets of DE miRNAs. The integrative analysis between miRNAs and mRNAs demonstrates the regulations of miRNAs on nearly four thousand differentially expressed genes and most of the biological pathways enriched in macrophage polarization. In summary, this study elucidates the expression profiles of miRNAs and their potential targetomes during macrophage polarization.

Figures

Figure 1
Figure 1. Validation of gene expression of four markers for macrophage polarization.
Bar plot of marker gene expression fold change at 4 time points in M1 and M2 macrophages, based on qRT-PCR (gray) and or averaged RNA-seq (black) results. A–D: TNF-α, IL-1β, Arg1 and CD206 expression, respectively.
Figure 2
Figure 2. Differentially expressed (DE) miRNAs in mouse M1 vs. M2 macrophages.
(A) log2 averaged fold-change of differentially expressed miRNAs at 4 time points, with highlights on a few miRNAs with the most drastic changes over time. (B) Hierarchical clustering among DE miRNAs based on Spearman’s correlation coefficient (SCC). The three less-studied miRNAs are highlighted by red. (C) qPCR validation of top 4 differentially expressed miRNAs for M1 and M2 conditions respectively, at 2 and 4 hours. (n = 3, *p < 0.05, **p < 0.001).
Figure 3
Figure 3. Differentially expressed (DE) genes in mouse M1 vs. M2 macrophages.
(A) Principal Component Analysis (PCA) plot of 18 samples based on all detected genes in RNA-seq (B) Venn diagram of DE genes at 4 time points. (C) The time-series trends of 4 clusters of DE genes, where each line represents the fold change relative to M0 state (inactivated macrophage at 0 hr) of one gene. Red color indicates stronger membership and green/blue color denotes weaker membership, as seen in the legend. Heatmap plot based on membership values was seen in Fig. S2. The enriched pathways in each cluster can be seen in Table S7.
Figure 4
Figure 4. The network of DE miRNAs and leading genes targeted by each DE miRNA.
Gene Set Enrichment Analysis (GSEA) was performed on the predicted targets of each miRNA, in order to obtain the leading genes of the significant (P < 0.05) pathways targeted by that particular miRNA. The color labels are: red (M1-specific miRNAs), green (M2-specific miRNAs), blue (DE and leading edge genes) and gray (non-DE and leading edge genes). The node size is proportional to the average fold change of M1 vs. M2 macrophages. The node shapes of diamond and circle denote transcriptional factors (TFs) and other non-TFs genes, respectively.
Figure 5
Figure 5. Heatmap to show the global enrichment of miRNA targets among DE genes.
For each time point, hypergeometric tests were performed to verify if the miRNA targets are indeed enriched among the DE genes, and the Benjamin-Hochberg(BH) adjusted p-value is then log(0.05) transformed per miRNA per time-point. Red indicates statistical significance, and blue indicates none. The miRNAs are clustered to demonstrate their relationships.
Figure 6
Figure 6. Highly-correlated M1- and M2-specific miRNA-target network.
The miRNAs with higher expression levels in M1 (A) and M2 (B) macrophages, are shown with their highly correlated targets (SCC > 0.8) that are predicted by targetScan. (A) Highly correlated miRNAs and targets in M1 macrophages. (B) Highly correlated miRNAs and targets in M2 macrophages. (C) Highly correlated miRNAs and C2H2 zinc-finger families member targets in both M1 and M2 macrophages. The node shape and color labels are the same as Fig. 4.
Figure 7
Figure 7. Validation of 3 new miRNAs in macrophage polarization.
(A–C) qRT-PCR validation for the expression of the three miRNAs miR-1931 (A), miR-3473e (B), miR-5128 (C), using primary BMDM polarized to the M1- or M2- phenotypes after 1, 2, 4, or 8 hours. (D,E) BMDM transfected with miRCURY LNA microRNA inhibitors or mimics of miR-5128 (100 μM) for 24 hours followed by M1 polarization for 2 hours or M2 polarization for 4 hours were analyzed by qRT-PCR for M2-associated gene expression. Changes in Arginase 1 (Arg1) (D) and Interleukin-10 (IL-10) (E) transcript levels are displayed as a percentage of M1- or M2-polarized, scrambled control-transfected cells. (n = 3, *p < 0.05, **p < 0.001).

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