We investigated genome-wide miRNA, mRNA and protein expression following human T-lymphocyte activation. T-lymphocyte activation relies on signaling cascades that create a balance between activation, memory and quiescence. This balance is modulated by mechanisms regulating gene expression including post-transcriptional miRNA regulation. Here we show a unique miRNA signature with a total of 71 differentially expressed miRNAs with 51 upregulated between 0 and 48 hours. This signature comprises 57 miRNAs with no documented roles in T-lymphocyte function. Additionally, our data validated previous findings for a number of miRNAs with known functions in T-lymphocytes: upregulation of miR-155 can regulate the susceptibility of human and murine CD4+
T cells to nTreg cell-mediated suppression (26
), the miR-17~92 cluster inhibits T cell activation (30
), miR-106a is implicated in IL-10 regulation(31
), miR-24 can inhibit cell proliferation by targeting cell-cycle genes(6
), and miR-21, upregulated by STAT3, prevents CD4+
T cell apoptosis and is implicated in lymphocyte oncogenesis (18
). Also, consistent with the importance of activation-induced miRNA expression, we observed upregulation of 10 miRNA biogenesis/processing machinery genes.
MiRNAs are known inhibitors of gene expression. The challenge is to map miRNAs to specific gene targets and the molecular networks they regulate. To address this challenge we investigated the predictive values of 4 widely used computational algorithms. First, if the results from all the algorithms are compared using a single miRNA hit/seed approach, the predicted targets are poorly correlated between methods to the extent that different methods will report very different results. Second, single hit/seed predictions did not correlate with mRNA repression. In contrast, combinatorial targeting (multiple seeds per target) gave the best predictions. Targetscan conservation predictions with combinatorial binding of 4 or more miRNAs showed the correlation between increased miRNA binding with decreased target gene expression.
By integrating activation-induced miRNA, mRNA and protein expression changes with target predictions, we tested our hypothesis that target genes are involved in regulating immune activation, cell proliferation and survival. Indeed, functional analysis demonstrated that downregulated miRNA targets populated signaling pathways highly enriched for immune response, proliferation, and survival. In contrast, activation-induced genes not predicted to be miRNA targets demonstrated significant enrichment for pathways of metabolism, DNA stability and cell cycle. These novel results reveal that predicted targets of activation-induced miRNAs are functionally distinct from non-target genes and presumably evolved with distinct selection pressures for such regulation. We also hypothesized that some targets might be specifically regulated by post-transcriptional mechanisms not coupled to mRNA decay. Based on our proteomics, we detected a number of such downregulated protein targets despite increased mRNA expression. Thus, inhibition of protein translation is not always coupled to corresponding mRNA degradation.
By investigating connectivity between predicted targets, we identified highly connected genes that function as network nodes, with closely connected genes being functionally similar. The top nodal gene PIK3R1 of one such network is a predicted target of miR-221 and 155, and downregulated at both mRNA and protein levels. This gene is an adaptor kinase involved in TCR signaling and CD28 co-stimulation, and regulates cell growth, proliferation, and T cell cytokine production (75
). Thus, functional network analysis underlines the value of the mapping done here based on global gene, protein and miRNA expression.
In validation, we focused on the functional roles of two top upregulated miRNAs in our data: miR-155, widely studied in T cells, and miR-221, not previously associated with T cell function. Knockdown of either miRNA produced a significant increase in proliferation of activated CD4+
T cells, confirming that these two miRNAs actually have anti-proliferative roles during activation. We identified 4 potential targets of miR-155 and/or 221, and mapped a functional network critical to T cell activation (). In addition to identifying PIK3R1 as a new target gene for miR-155 and 221, we discovered that the transcription factor FOS is also a target of both miR-155 and 221. We identified two more targets of miR-155: the novel IRS2, an adaptor for tyrosine kinases, and a previously verified miR-155 target IKBKE, that regulates NF-κB activation (83
). Knockdown of miR-221 and/or 155 increased target mRNA expression for PIK3R1, FOS, and IRS2.
In conclusion, we propose a model where in the course of T-lymphocyte activation by TCR engagement and CD28 co-stimulation, there is a significant upregulation of miRNAs that are critical to this process. These activation-induced miRNAs create a negative-feedback loop to inhibit cell proliferation and regulate cell survival by targeting a series of molecular networks that we have mapped. In parallel, there is also a subset of miRNAs downregulated by activation and 84% of their predicted target genes are shown to be upregulated at the mRNA level. Moreover, there is a functionally-specific class of genes linked closely to the immune response that evolved to be the natural targets of these miRNAs in T-lymphocytes, with functions revealed by molecular networking mapping that are clearly distinct from the activation-induced genes that are not miRNA targets.