Teresa Colombo

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.

Computational analysis identifies a sponge interaction network between long non-coding RNAs and messenger RNAs in human breast cancer

BackgroundNon-coding RNAs (ncRNAs) are emerging as key regulators of many cellular processes in both physiological and pathological states. Moreover, the constant discovery of new non-coding RNA species suggests that the study of their complex functions is still in its very early stages. This variegated class of RNA species encompasses the well-known microRNAs (miRNAs) and the most recently acknowledged long non-coding RNAs (lncRNAs). Interestingly, in the last couple of years, a few studies have shown that some lncRNAs can act as miRNA sponges, i.e. as competing endogenous RNAs (ceRNAs), able to reduce the amount of miRNAs available to target messenger RNAs (mRNAs).ResultsWe propose a computational approach to explore the ability of lncRNAs to act as ceRNAs by protecting mRNAs from miRNA repression. A seed match analysis was performed to validate the underlying regression model. We built normal and cancer networks of miRNA-mediated sponge interactions (MMI-networks) using breast cancer expression data provided by The Cancer Genome Atlas.ConclusionsOur study highlights a marked rewiring in the ceRNA program between normal and pathological breast tissue, documented by its “on/off” switch from normal to cancer, and vice-versa. This mutually exclusive activation confers an interesting character to ceRNAs as potential oncosuppressive, or oncogenic, protagonists in cancer. At the heart of this phenomenon is the lncRNA PVT1, as illustrated by both the width of its antagonist mRNAs in normal-MMI-network, and the relevance of the latter in breast cancer. Interestingly, PVT1 revealed a net binding preference towards the mir-200 family as the bone of contention with its rival mRNAs.

MicroRNAs Cooperatively Inhibit a Network of Tumor Suppressor Genes to Promote Pancreatic Tumor Growth and Progression

BACKGROUND & AIMS There has not been a broad analysis of the combined effects of altered activities of microRNAs (miRNAs) in pancreatic ductal adenocarcinoma (PDAC) cells, and it is unclear how these might affect tumor progression or patient outcomes. METHODS We combined data from miRNA and messenger RNA (mRNA) expression profiles and bioinformatic analyses to identify an miRNA-mRNA regulatory network in PDAC cell lines (PANC-1 and MIA PaCa-2) and in PDAC samples from patients. We used this information to identify miRNAs that contribute most to tumorigenesis. RESULTS We identified 3 miRNAs (MIR21, MIR23A, and MIR27A) that acted as cooperative repressors of a network of tumor suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of MIR21, MIR23A, and MIR27A had synergistic effects in reducing proliferation of PDAC cells in culture and growth of xenograft tumors in mice. The level of inhibition was greater than that of inhibition of MIR21 alone. In 91 PDAC samples from patients, high levels of a combination of MIR21, MIR23A, and MIR27A were associated with shorter survival times after surgical resection. CONCLUSIONS In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to growth of PDACs. These findings indicate that miRNAs act together to promote tumor progression; therapeutic strategies might require inhibition of several miRNAs.

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.

Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression

Caenorhabditis elegans is one of the most prominent model systems for embryogenesis, but collecting many precisely staged embryos has been impractical. Thus, early C. elegans embryogenesis has not been amenable to most high-throughput genomics or biochemistry assays. To overcome this problem, we devised a method to collect staged C. elegans embryos by fluorescence-activated cell sorting (eFACS). In a proof-of-principle experiment, we found that a single eFACS run routinely yielded tens of thousands of almost perfectly staged 1-cell stage embryos. As the earliest embryonic events are driven by posttranscriptional regulation, we combined eFACS with second-generation sequencing to profile the embryonic expression of small, noncoding RNAs. We discovered complex and orchestrated changes in the expression between and within almost all classes of small RNAs, including microRNAs and 26G-RNAs, during embryogenesis.

PVT1: A Rising Star among Oncogenic Long Noncoding RNAs

It is becoming increasingly clear that short and long noncoding RNAs critically participate in the regulation of cell growth, differentiation, and (mis)function. However, while the functional characterization of short non-coding RNAs has been reaching maturity, there is still a paucity of well characterized long noncoding RNAs, even though large studies in recent years are rapidly increasing the number of annotated ones. The long noncoding RNA PVT1 is encoded by a gene that has been long known since it resides in the well-known cancer risk region 8q24. However, a couple of accidental concurrent conditions have slowed down the study of this gene, that is, a preconception on the primacy of the protein-coding over noncoding RNAs and the prevalent interest in its neighbor MYC oncogene. Recent studies have brought PVT1 under the spotlight suggesting interesting models of functioning, such as competing endogenous RNA activity and regulation of protein stability of important oncogenes, primarily of the MYC oncogene. Despite some advancements in modelling the PVT1 role in cancer, there are many questions that remain unanswered concerning the precise molecular mechanisms underlying its functioning.

Growth arrest-specific transcript 5 associated snoRNA levels are related to p53 expression and DNA damage in colorectal cancer

Background The growth arrest-specific transcript 5 gene (GAS5) encodes a long noncoding RNA (lncRNA) and hosts a number of small nucleolar RNAs (snoRNAs) that have recently been implicated in multiple cellular processes and cancer. Here, we investigate the relationship between DNA damage, p53, and the GAS5 snoRNAs to gain further insight into the potential role of this locus in cell survival and oncogenesis both in vivo and in vitro. Methods We used quantitative techniques to analyse the effect of DNA damage on GAS5 snoRNA expression and to assess the relationship between p53 and the GAS5 snoRNAs in cancer cell lines and in normal, pre-malignant, and malignant human colorectal tissue and used biological techniques to suggest potential roles for these snoRNAs in the DNA damage response. Results GAS5-derived snoRNA expression was induced by DNA damage in a p53-dependent manner in colorectal cancer cell lines and their levels were not affected by DICER. Furthermore, p53 levels strongly correlated with GAS5-derived snoRNA expression in colorectal tissue. Conclusions In aggregate, these data suggest that the GAS5-derived snoRNAs are under control of p53 and that they have an important role in mediating the p53 response to DNA damage, which may not relate to their function in the ribosome. We suggest that these snoRNAs are not processed by DICER to form smaller snoRNA-derived RNAs with microRNA (miRNA)-like functions, but their precise role requires further evaluation. Furthermore, since GAS5 host snoRNAs are often used as endogenous controls in qPCR quantifications we show that their use as housekeeping genes in DNA damage experiments can lead to inaccurate results.

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.

Increased chronic lymphocytic leukemia proliferation upon IgM stimulation is sustained by the upregulation of miR-132 and miR-212

To assess the involvement of microRNAs (miRNAs) in B‐cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross‐linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR‐132 and miR‐212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR‐132 and miR‐212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR‐132 and miR‐212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR‐132 and miR‐212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention.

The p53 miRNA interactome and its potential role in the cancer clinic

p53 is one of the most frequently mutated tumor suppressors. It regulates protein-coding genes and noncoding RNAs involved in many cellular processes, functioning predominantly at the transcriptional level but also through nontranscriptional processes. miRNAs have recently been identified as key mediators of the p53 stress-response pathway. p53 regulates miRNA transcription and processing, and miRNAs regulate p53 activity and expression and, accordingly, various feedback/feed-forward loops have been identified. Many chemotherapeutic agents induce cancer cell death or senescence via DNA damage and the subsequent activation of p53. Resistance to chemotherapy can occur due to the mutation of components in p53 signaling networks. A better understanding of the role of the various components within these pathways and their interactions with each other may allow the modification and improvement of current treatments, and the design of novel therapies. Improving our knowledge of the role of miRNAs in such p53 signaling networks may be crucial to achieving this.