Dynamic nature of noncoding RNA regulation of adaptive immune response.

Immune response plays a fundamental role in protecting the organism from infections; however, dysregulation often occurs and can be detrimental for the organism, leading to a variety of immune-mediated diseases. Recently our understanding of the molecular and cellular networks regulating the immune response, and, in particular, adaptive immunity, has improved dramatically. For many years, much of the focus has been on the study of protein regulators; nevertheless, recent evidence points to a fundamental role for specific classes of noncoding RNAs (ncRNAs) in regulating development, activation and homeostasis of the immune system. Although microRNAs (miRNAs) are the most comprehensive and well-studied, a number of reports suggest the exciting possibility that long ncRNAs (lncRNAs) could mediate host response and immune function. Finally, evidence is also accumulating that suggests a role for miRNAs and other small ncRNAs in autocrine, paracrine and exocrine signaling events, thus highlighting an elaborate network of regulatory interactions mediated by different classes of ncRNAs during immune response. This review will explore the multifaceted roles of ncRNAs in the adaptive immune response. In particular, we will focus on the well-established role of miRNAs and on the emerging role of lncRNAs and circulating ncRNAs, which all make indispensable contributions to the understanding of the multilayered modulation of the adaptive immune response.

An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes.

Activation of the T cell-mediated immune response has been associated with changes in the expression of specific microRNAs (miRNAs). However, the role of miRNAs in the development of an effective immune response is just beginning to be explored. This study focuses on the functional role of miR-146a in T lymphocyte-mediated immune response and provides interesting clues on the transcriptional regulation of miR-146a during T-cell activation. We show that miR-146a is low in human naive T cells and is abundantly expressed in human memory T cells; consistently, miR-146a is induced in human primary T lymphocytes upon T-cell receptor (TCR) stimulation. Moreover, we identified NF-kB and c-ETS binding sites as required for the induction of miR-146a transcription upon TCR engagement. Our results demonstrate that several signaling pathways, other than inflammation, are influenced by miR-146a. In particular, we provide experimental evidence that miR-146a modulates activation-induced cell death (AICD), acting as an antiapoptotic factor, and that Fas-associated death domain (FADD) is a target of miR-146a. Furthermore, miR-146a enforced expression impairs both activator protein 1 (AP-1) activity and interleukin-2 (IL-2) production induced by TCR engagement, thus suggesting a role of this miRNA in the modulation of adaptive immunity.

BCR ligation induced by IgM stimulation results in gene expression and functional changes only in IgV H unmutated chronic lymphocytic leukemia (CLL) cells.

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.

Characterization of B- and T-lineage acute lymphoblastic leukemia by integrated analysis of MicroRNA and mRNA expression profiles.

Acute lymphoblastic leukemia (ALL) is an heterogeneous disease comprising several subentities that differ for both immunophenotypic and molecular characteristics. Over the years, the biological understanding of this neoplasm has largely increased. Gene expression profiling has allowed to identify specific signatures for the different ALL subsets and permitted the identification of pathways deregulated by a given lesion. MicroRNAs (miRNAs) are small noncoding RNAs, which play a pivotal role in several cellular functions. In this study, we investigated miRNAs expression profiles in a series of adult ALL cases by microarray analysis. Unsupervised hierarchical clustering largely recapitulated ALL subgroups. Furthermore, we identified miR-148, miR-151, and miR-424 as discriminative of T-lineage versus B-lineage ALL; ANOVA highlighted a set of six miRNAs-namely miR-425-5p, miR-191, miR-146b, miR-128, miR-629, and miR-126-that can discriminate B-lineage ALL subgroups harboring specific molecular lesions. These results were confirmed and extended by quantitative-PCR on a further cohort of cases. Finally, we used Pearson correlation analysis to combine miRNA and gene expression profiles. The distribution of correlation coefficients generated by comparing the expression of every miRNA/gene pair in our data set shows enrichment of both positively and negatively correlated pairs over background distributions obtained using randomized data. Moreover, a clear enrichment for predicted miRNA:target pairs is observed at negative correlation coefficient intervals. Signal-to-noise ratio highlighted several miRNA/gene pairs with a possible role in the disease. In fact, gene set enrichment analysis of genes composing the selected miRNA/gene pairs displays over-representation of functional categories related to cancer and cell-cycle regulation.

Functional Roles and Therapeutic Applications of Exosomes in Hepatocellular Carcinoma.

Exosomes are important in intercellular communication. They assure the horizontal transfer of specific functional contents (i.e., proteins, lipids, RNA molecules, and circulating DNA) from donor to recipient cells. Notably, tumor-derived exosomes (TDEs) appear to be an important vehicle of specific signals in cancer, impacting on tumor growth and metastasis. Recent researches point to the characterization of exosomes in Hepatocellular Carcinoma (HCC), the major adult liver malignancy. In this review, we summarize current findings on HCC exosomes, focusing on the identification of noncoding RNAs as exosome-enriched functional regulators and new potential biomarkers. The great potential of exosomes in future HCC diagnostic and therapeutic approaches is underlined.

TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

Epigenetic regulation in hepatocellular carcinoma requires long noncoding RNAs.

Recent evidence has proven the relevance of epigenetic changes in the development of hepatocellular carcinoma (HCC), the major adult liver malignancy. Moreover, HCC onset and progression correlate with the deregulation of several long noncoding RNAs (lncRNAs), exhibiting great biological significance. As discussed in this review, many of these transcripts are able to specifically act as tumor suppressors or oncogenes by means of their role as molecular platforms. Indeed, these lncRNAs are able to bind and recruit epigenetic modifiers on specific genomic loci, ultimately resulting in regulation of the gene expression relevant in cancer development. The evidence presented in this review highlights that lncRNAs-mediated epigenetic regulation should be taken into account for potential targeted therapeutic approaches.

miR-21 is a negative modulator of T-cell activation.

microRNAs (miRNAs) are a class of small non-coding RNAs acting as post-transcriptional regulators of gene expression and play fundamental roles in regulating immune response and autoimmunity. We show that memory T-lymphocytes express higher levels of miR-21 compared to naïve T-lymphocytes and that miR-21 expression is induced upon TCR engagement of naïve T-cells. We identify bona fide miR-21 targets by direct immuno-purification and profiling of AGO2-associated mRNAs in Jurkat cells over-expressing miR-21. Our analysis shows that, in T-lymphocytes, miR-21 targets genes are involved in signal transduction. Coherently, TCR signalling is dampened upon miR-21 over-expression in Jurkat cells, resulting in lower ERK phosphorylation, AP-1 activation and CD69 expression. Primary human lymphocytes in which we impaired miR-21 activity, display IFN-γ production enhancement and stronger activation in response to TCR engagement as assessed by CD69, OX40, CD25 and CD127 analysis. By intracellular staining of the endogenous protein in primary T-lymphocytes we validate three key regulators of lymphocyte activation as novel miR-21 targets. Our results highlight an unexpected function of miR-21 as a negative modulator of signal transduction downstream of TCR in T-lymphocytes.

miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis.

miRNAs have recently emerged as key regulators of the immune system, being involved in lymphocyte selection and proliferation, in T(reg) cells differentiation, and in hematopoiesis in general. Rheumatoid arthritis (RA) is an autoimmune pathology the etiology of which is still obscure. Although a multifactorial pathogenesis has been hypothesized, the precise mechanisms leading to the disease are still poorly understood at the molecular level. miRNA expression profile analysis highlighted that miR-223 is the only miRNA that is strikingly deregulated in peripheral T-lymphocytes from RA patients compared with healthy donors. Further analysis by quantitative reverse transcription-polymerase chain analysis confirmed that miR-223 is overexpressed in T-lymphocytes from RA patients (n = 28) compared with healthy donors (n = 10). Moreover, purification of different T-lymphocyte populations from RA patients highlights that miR-223 is expressed at higher levels in naive CD4(+) lymphocytes, whereas its expression is barely detectable in T(h)-17 cells. In summary, our data provide a first characterization of the miRNA expression profiles of peripheral T-lymphocytes of RA patients, identifying miR-223 as overexpressed in CD4(+) naive T-lymphocytes from these individuals. A deeper analysis of the biologic functions and effects of the expression of miR-223 in T-lymphocytes is needed to clarify the exact link between our observation and the disease.

TGFbeta-induced EMT requires focal adhesion kinase (FAK) signaling.

The epithelial-to-mesenchymal transition (EMT) is a crucial process, occurring both during development and tumor progression, by which an epithelial cell undergoes a conversion to a mesenchymal phenotype, dissociates from initial contacts and migrates to secondary sites. We recently reported that in hepatocytes the multifunctional cytokine TGFbeta induces a full EMT characterized by (i) Snail induction, (ii) E-cadherin delocalization and down-regulation, (iii) down-regulation of the hepatocyte transcriptional factor HNF4alpha and (iv) up-regulation of mesenchymal and invasiveness markers. In particular, we showed that Snail directly causes the transcriptional down-regulation of E-cadherin and HNF4, while it is not sufficient for the up-regulation of mesenchymal and invasiveness EMT markers. In this paper, we show that in hepatocytes TGFbeta induces a Src-dependent activation of the focal adhesion protein FAK. More relevantly, we gathered results indicating that FAK signaling is required for (i) transcriptional up-regulation of mesenchymal and invasiveness markers and (ii) delocalization of membrane-bound E-cadherin. Our results provide the first evidence of FAK functional role in TGFbeta-mediated EMT in hepatocytes.

Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia.

MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of genes at the posttranscriptional level. These small molecules have been shown to be involved in cancer, apoptosis, and cell metabolism. In the present study we provide an informative profile of the expression of miRNAs in primary chronic lymphocytic leukemia (CLL) cells using 2 independent and quantitative methods: miRNA cloning and quantitative real-time-polymerase chain reaction (qRT-PCR) of mature miRNAs. Both approaches show that miR-21 and miR-155 are dramatically overexpressed in patients with CLL, although the corresponding genomic loci are not amplified. miR-150 and miR-92 are also significantly deregulated in patients with CLL. In addition, we detected a marked miR-15a and miR-16 decrease in about 11% of cases. Finally, we identified a set of miRNAs whose expression correlates with biologic parameters of prognostic relevance, particularly with the mutational status of the IgV(H) genes. In summary, the results of this study offer for the first time a comprehensive and quantitative profile of miRNA expression in CLL and their healthy counterpart, suggesting that miRNAs could play a primary role in the disease itself.