Microguards and micromessengers of the genome

The regulation of gene expression is of fundamental importance to maintain organismal function and integrity and requires a multifaceted and highly ordered sequence of events. The cyclic nature of gene expression is known as ‘transcription dynamics’. Disruption or perturbation of these dynamics can result in significant fitness costs arising from genome instability, accelerated ageing and disease. We review recent research that supports the idea that an important new role for small RNAs, particularly microRNAs (miRNAs), is in protecting the genome against short-term transcriptional fluctuations, in a process we term ‘microguarding’. An additional emerging role for miRNAs is as ‘micromessengers’—through alteration of gene expression in target cells to which they are trafficked within microvesicles. We describe the scant but emerging evidence that miRNAs can be moved between different cells, individuals and even species, to exert biologically significant responses. With these two new roles, miRNAs have the potential to protect against deleterious gene expression variation from perturbation and to themselves perturb the expression of genes in target cells. These interactions between cells will frequently be subject to conflicts of interest when they occur between unrelated cells that lack a coincidence of fitness interests. Hence, there is the potential for miRNAs to represent both a means to resolve conflicts of interest, as well as instigate them. We conclude by exploring this conflict hypothesis, by describing some of the initial evidence consistent with it and proposing new ideas for future research into this exciting topic

MicroRNA profiling reveals that miR-21, miR486 and miR-214 are upregulated and involved in cell survival in Sézary syndrome

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma and its pathogenesis is still unknown. Diagnosis/prognosis may strongly ameliorate the management of SS individuals. Here, we profiled the expression of 470 microRNAs (miRNAs) in a cohort of 22 SS patients, and we identified 45 miRNAs differentially expressed between SS and controls. Using predictive analysis, a list of 19 miRNAs, including miR-21, miR-214, miR-486, miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs including again miR-21, potentially able to discriminate patients with unfavorable and favorable outcome. We validated our data for miR-21, miR-214 and miR-486 by qRT-PCR, including an additional set of array-independent SS cases. In addition, we also provide an in vitro evidence for a contribution of miR-214, miR-486 and miR-21 to apoptotic resistance of CTCL cell line

Restoration of Altered MicroRNA Expression in the Ischemic Heart with Resveratrol

Resveratrol, a constituent of red wine, is important for cardioprotection. MicroRNAs are known regulators for genes involved in resveratrol-mediated cardiac remodeling and the regulatory pathway involving microRNA has not been studied so far.We explored the cardioprotection by resveratrol in ischemia/reperfusion model of rat and determined cardiac functions. miRNA profile was determined from isolated RNA using quantitative Real-time PCR based array. Systemic analyses of miRNA array and theirs targets were determined using a number of computational approaches.Cardioprotection by resveratrol and its derivative in ischemia/reperfusion [I/R] rat model was examined with miRNA expression profile. Unique expression pattern were found for each sample, particularly with resveratrol [pure compound] and longevinex [commercial resveratrol formulation] pretreated hearts. Longevinex and resveratrol pretreatment modulates the expression pattern of miRNAs close to the control level based on PCA analyses. Differential expression was observed in over 25 miRNAs, some of them, such as miR-21 were previously implicated in cardiac remodeling. The target genes for the differentially expressed miRNA include genes of various molecular function such as metal ion binding, sodium-potassium ion, transcription factors, which may play key role in reducing I/R injury.Rats pretreated with resveratrol for 3 weeks leads to significant cardioprotection against ischemia/reperfusion injury. A unique signature of miRNA profile is observed in control heart pretreated with resveratrol or longevinex. We have determined specific group of miRNA in heart that have altered during IR injuries. Most of those altered microRNA expressions modulated close to their basal level in resveratrol or longevinex treated I/R mice

MicroRNA Fingerprints Identify miR-150 as a Plasma Prognostic Marker in Patients with Sepsis

BACKGROUND: The physiopathology of sepsis continues to be poorly understood, and despite recent advances in its management, sepsis is still a life-threatening condition with a poor outcome. If new diagnostic markers related to sepsis pathogenesis will be identified, new specific therapies might be developed and mortality reduced. Small regulatory non-coding RNAs, microRNAs (miRNAs), were recently linked to various diseases; the aim of our prospective study was to identify miRNAs that can differentiate patients with early-stage sepsis from healthy controls and to determine if miRNA levels correlate with the severity assessed by the Sequential Organ Failure Assessment (SOFA) score. METHODOLOGY/PRINCIPAL FINDINGS: By using genome-wide miRNA profiling by microarray in peripheral blood leukocytes, we found that miR-150, miR-182, miR-342-5p, and miR-486 expression profiles differentiated sepsis patients from healthy controls. We also proved by quantitative reverse transcription-polymerase chain reaction that miR-150 levels were significantly reduced in plasma samples of sepsis patients and correlated with the level of disease severity measured by the SOFA score, but were independent of the white blood counts (WBC). We found that plasma levels of tumor necrosis factor alpha, interleukin-10, and interleukin-18, all genes with sequence complementarity to miR-150, were negatively correlated with the plasma levels of this miRNA. Furthermore, we identified that the plasma levels ratio for miR-150/interleukin-18 can be used for assessing the severity of the sepsis. CONCLUSIONS/SIGNIFICANCE: We propose that miR-150 levels in both leukocytes and plasma correlate with the aggressiveness of sepsis and can be used as a marker of early sepsis. Furthermore, we envision miR-150 restoration as a future therapeutic option in sepsis patients

MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

BACKGROUND & AIM: MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis. METHODS: Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed. RESULTS: MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor kappa beta (NF-kappaB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and alpha smooth muscle actin (alphaSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-beta (TGFbeta) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein beta (C/EBPbeta) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice. CONCLUSIONS: Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis

Induction of IL-4R alpha-dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4-driven murine macrophage proliferation in vivo

Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4–receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4–driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα–induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4–driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings

The Expression of Vasoactive Intestinal Peptide Receptor 1 Is Negatively Modulated by MicroRNA 525-5p

Background: The human Vasoactive Intestinal Peptide (VIP) is a neurokine with effects on the immune system where it is involved in promoting tolerance. In this context, one of its receptors, VPAC1, has been found to be down-modulated in cells of the immune network in response to activating stimuli. In particular, the bacterial liposaccaride (LPS), a strong activator of the innate immune system, induces a rapid decrease of VPAC1 expression in monocytes and this event correlates with polymorphisms in the 3'-UTR of the gene. Methodology/Principal Findings: MicroRNA 525-5p, having as putative target the 3'-UTR region of VPAC1, has been analysed for its expression in monocytes and for its role in down-modulating VPAC1 expression. We report here that miR-525-5p is promptly up-regulated in LPS-treated monocytes. This microRNA, when co-transfected in 293T cells together with a construct containing the 3'-UTR of the VPAC1 gene, significantly reduced the luciferase activity in a standard expression assay. The U937 cell line as well as primary monocytes enforced to express miR-525-5p, both down-modulate VPAC1 expression at similar extent. Conclusions/Significance: Our results show that the response to an inflammatory stimulus elicits in monocytes a rapid increase of miR-525-5p that targets a signaling pathway involved in the control of the immune homeostasis

Analysis of microRNA turnover in mammalian cells following Dicer1 ablation

Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function

Selective inhibition of microRNA accessibility by RBM38 is required for p53 activity

MicroRNAs (miRNAs) interact with 3′-untranslated regions of messenger RNAs to restrict expression of most protein-coding genes during normal development and cancer. RNA-binding proteins (RBPs) can control the biogenesis, stability and activity of miRNAs. Here we identify RBM38 in a genetic screen for RBPs whose expression controls miRNA access to target mRNAs. RBM38 is induced by p53 and its ability to modulate miRNA-mediated repression is required for proper p53 function. In contrast, RBM38 shows lower propensity to block the action of the p53-controlled miR-34a on SIRT1. Target selectivity is determined by the interaction of RBM38 with uridine-rich regions near miRNA target sequences. Furthermore, in large cohorts of human breast cancer, reduced RBM38 expression by promoter hypermethylation correlates with wild-type p53 status. Thus, our results indicate a novel layer of p53 gene regulation, which is required for its tumour suppressive function

Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines