Identification of microRNA-regulated gene networks by expression analysis of target genes

Abstract

MicroRNAs (miRNAs) and transcription factors control eukaryotic cell proliferation, differentiation, and metabolism through their specific gene regulatory networks. However, differently from transcription factors, our understanding of the processes regulated by miRNAs is currently limited. Here, we introduce gene network analysis as a new means for gaining insight into miRNA biology. A systematic analysis of all human miRNAs based on Co-expression Meta-analysis of miRNA Targets (CoMeTa) assigns high-resolution biological functions to miRNAs and provides a comprehensive, genome-scale analysis of human miRNA regulatory networks. Moreover, gene cotargeting analyses show that miRNAs synergistically regulate cohorts of genes that participate in similar processes. We experimentally validate the CoMeTa procedure through focusing on three poorly characterized miRNAs, miR-519d/190/340, which CoMeTa predicts to be associated with the TGFβ pathway. Using lung adenocarcinoma A549 cells as a model system, we show that miR-519d and miR-190 inhibit, while miR-340 enhances TGFβ signaling and its effects on cell proliferation, morphology, and scattering. Based on these findings, we formalize and propose co-expression analysis as a general paradigm for second-generation procedures to recognize bona fide targets and infer biological roles and network communities of miRNAs.

Figure 1.

The CoMeTa procedure. (A) For simplicity, the strategy is described on a subgroup of 10 genes. Multiple transcriptional controls (arrows) for these genes are shown, including a specific miRNA (miRNA-X, blue arrows). In the example, Gene 8 (yellow box) and Gene 9 (red box) are, respectively, a false positive and a false negative result of sequence-based software predictions, used to seed the analysis. In co-expression lists, the ranking of the genes is an index of their expression correlations with the probe gene. A co-rank list is obtained by averaging the co-expression lists. The false-positive target ranks low in the co-rank list, whereas the false-negative target ranks high and can be identified by subsequent de novo sequence analyses (additional targets [AT] list). The CoMeTa output consists of the list of predicted targets ranked by expression analysis. Co-expression lists are subsequently used for further analysis (see text). (B) Distribution of known miRNA targets (horizontal lines in the top panel) within CoMeTa lists, for three independent data sets of miRNA targets (DS1, DS2, DS3). Counts within the first 30^th and 50^th percentiles are provided for each data set in the lower table. The average number of targets for each miRNA present in the first 50^th percentile of CoMeTa lists is 750 (DS1), 850 (DS2), and 900 (DS3). P-values: <10⁻³⁹ (DS1), <10⁻²⁹ (DS2), and <10⁻²³ (DS3).

Figure 2.

COOL analysis of miRNA-predicted target transcriptional networks. (A) Kernel density estimation of R² values for the normal probability plot analysis of miRNA clusters (red line) and size-matched random clusters (blue line). An R² value of 0.912 represents the lowest value for the random clusters, and this was used as the threshold (green dotted line) to select the significant miRNA clusters. (Black bars) R² values of DS1-associated clusters. (B,C) COOL heat-maps (left) and their enrichment plots generated by gene-set enrichment analysis (right) for the data set of probes differentially expressed after miR-26b transfection (B) and miR-98 transfection (C). (Blue lines in the plots) Enrichment scores; (vertical black bars below the plots) positions of the probes from the analyzed COOL clusters according to the ranking of their expression levels, i.e., from the most down-regulated (left) to the most up-regulated. (ES) enrichment score; (FDR) false discovery rate. (D) Frequency distribution of functions assigned to COOL clusters as grouped in macrocategories. The number of miRNAs associated with each macrocategory is shown in parenthesis.

Figure 3.

Overview of COOL clusters with known and predicted functions. Analyses of miR-155 (A), miR-1 (B), miR-130a (C), miR-519d (D), miR-190 (E), and miR-340 (F). The graphs represent the COOL heat-maps of putative targets generated according to their reciprocal expression relationships. (Red and yellow boxes) Clusters with an R² value higher or lower than the set threshold, respectively. (PTs) Predicted targets. Some of the functional categories enriched in the significant miRNA clusters are indicated.

Figure 4.

miR-519d, miR-190, and miR-340 in the TGFβ signaling pathway. Schematic of the network of interactions between genes and proteins involved in TGFβ signaling. Putative targets of miR-519d (purple), miR-190 (orange), and miR-340 (yellow) are indicated. (Double vertical lines) The cell membrane; (dotted vertical line) the nuclear membrane; (dotted horizontal lines) links to other cellular pathways.

Figure 5.

miR-519d, miR-190, and miR-340 modulate TGFβ signaling. Analysis of cell proliferation, cell morphology, and cell scattering following miRNA transfection in A549 cells, with or without TGFB1 addition. All data and confocal microscope images are representative of at least three experiments for each miRNA. Cell proliferation is expressed in relative luminescence units (RLU), determined by luminescence-based cell viability assays. Confocal microscopy was performed after staining with FITC-phalloidin (green) and DAPI (blue). Cell scattering was quantified by digital image analysis of local cell density.

Figure 6.

miRNA community networks. (A) Graphical representation of the community organization of human miRNA downstream transcriptional networks. (Gray circles) miRNAs. miRNAs that belong to the same community are linked with edges of the same color. For each community an exemplar (center of the miRNA community) was chosen and is indicated with a blue triangle. (Red triangle) miRNAs that link together different communities. (B) Enlarged section from A, showing the composition of miRCo16 and the mutual relationships of its miRNAs and shared target genes. (C) Frequency distribution of functions assigned to miRCOs as grouped in macrocategories. The number of miRCOs associated with each macrocategory is shown in parenthesis.