MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.

/pdf/microrna-sponges-competitive-inhibitors-of-small-rnas-in-1wce8re8p1.pdf

MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells

The immune synapse is an exquisitely evolved means of communication between T cells and antigen-presenting cells (APCs) during antigen recognition. Recent evidence points to the transfer of RNA via exosomes as a novel mode of intercellular communication. Here we show that exosomes of T, B and dendritic immune cells contain microRNA (miRNA) repertoires that differ from those of their parent cells. We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation. Inhibition of exosome production by targeting neutral sphingomyelinase-2 impairs transfer of miRNAs to APCs. Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells. Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.

/pdf/unidirectional-transfer-of-microrna-loaded-exosomes-from-t-36wk685m03.pdf

Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells

Early studies have shown how aberrantly expressed microRNAs are a hallmark of several diseases like cancer. MicroRNA expression profiling was shown to be associated with tumour development, progression and response to therapy, suggesting their possible use as diagnostic, prognostic and predictive biomarkers. Moreover, based on the increasing number of studies demonstrating that microRNAs can function as potential oncogenes or oncosuppressor genes, with the goal to improve disease response and increase cure rates, miRNA-based anticancer therapies have recently been exploited, either alone or in combination with current targeted therapies. The advantage of using microRNA approaches is based on its ability to concurrently target multiple effectors of pathways involved in cell differentiation, proliferation and survival. Here, we review our current knowledge about the involvement of microRNAs in cancer, and their potential as diagnostic, prognostic and therapeutic tools.

https://www.embopress.org/doi/pdf/10.1002/emmm.201100209

MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review

Changes in the patterns of gene expression are widely believed to underlie many of the phenotypic differences within and between species. Although much emphasis has been placed on changes in transcriptional regulation, gene expression is regulated at many levels, all of which must ultimately be studied together to obtain a complete picture of the evolution of gene expression. Here we compare the evolution of transcriptional regulation and post-transcriptional regulation that is mediated by microRNAs, a large class of small, non-coding RNAs in plants and animals, focusing on the evolution of the individual regulators and their binding sites. As an initial step towards integrating these mechanisms into a unified framework, we propose a simple model that describes the transcriptional regulation of new microRNA genes.

http://materiais.dbio.uevora.pt/BD/Crescimento/TFs-miRNAs.pdf

The evolution of gene regulation by transcription factors and microRNAs

Mammalian microRNAs (miRNAs) have recently been identified as important regulators of gene expression, and they function by repressing specific target genes at the post-transcriptional level. Now, studies of miRNAs are resolving some unsolved issues in immunology. Recent studies have shown that miRNAs have unique expression profiles in cells of the innate and adaptive immune systems and have pivotal roles in the regulation of both cell development and function. Furthermore, when miRNAs are aberrantly expressed they can contribute to pathological conditions involving the immune system, such as cancer and autoimmunity; they have also been shown to be useful as diagnostic and prognostic indicators of disease type and severity. This Review discusses recent advances in our understanding of both the intended functions of miRNAs in managing immune cell biology and their pathological roles when their expression is dysregulated.

Physiological and pathological roles for microRNAs in the immune system

miR-181a is an intrinsic modulator of T cell sensitivity and selection

MicroRNAs (miRNAs) control the expression of protein-coding genes in normal hematopoietic cells and, consequently, aberrant expression may contribute to leukemogenesis. To identify miRNAs relevant to pediatric acute lymphoblastic leukemia (ALL), we cloned 105 known and 8 new miRNA genes expressed in patients' leukemia cells. Instead of known miRNA genes, new miRNA genes were not evolutionarily conserved. Quantification of 19 selected miRNA genes revealed an aberrant expression in ALL as compared with normal CD34+ cells (P <or= 0.02); both upregulated (14/19) and downregulated (5/19) expressions were observed. Eight miRNAs were differentially expressed between MLL and non-MLL precursor B-ALL cases (P<0.05). Most remarkably, miR-708 was 250- up to 6500-fold higher expressed in 57 TEL-AML1, BCR-ABL, E2A-PBX1, hyperdiploid and B-other cases than in 20 MLL-rearranged and 15 T-ALL cases (0.0001<P<0.01), whereas the expression of miR-196b was 500-fold higher in MLL-rearranged and 800-fold higher in 5 of 15 T-ALL cases as compared with the expression level in the remaining precursor B-ALL cases (P<0.001). The expression did not correlate with the maturation status of leukemia cells based on immunoglobulin and T-cell receptor rearrangements, immunophenotype or MLL-fusion partner. In conclusion, we identified new miRNA genes and showed that miRNA expression profiles are ALL subtype-specific rather than linked to the differentiation stadium associated with these subtypes.

Identification of new microRNA genes and aberrant microRNA profiles in childhood acute lymphoblastic leukemia.

MicroRNAs (miRNAs) are a diverse and abundant class of ˜22-nucleotide (nt) endogenous regulatory RNAs that play a variety of roles in animal cells by controlling gene expression at the posttranscriptional level Increased miR-181a expression in mature T cells is shown to cause a marked increase in T cell activation and augments T cell sensitivity to peptide antigens Moreover, T cell blasts with higher miR-181a expression become reactive to antagonists The effects of miR-181a on antigen discrimination are in part achieved by dampening the expression of multiple negative regulators in the T cell receptor (TCR) signaling pathway, including PTPN22 and the dual specificity phosphatases DUSP5 and DUSP6 This results in a reduction in the TCR signaling threshold, thus quantitatively and qualitatively enhancing T cell sensitivity to antigens

Modulation of T cell signaling threshold and T cell sensitivity to antigens

Les microARN (miARN) constituent une classe diverse et abondante d'ARN regulateurs endogenes d'environ 22 nucleotides (nt) revetant une pluralite de fonctions dans les cellules animales par regulation de l'expression genetique au niveau post-transcriptionnel. On a observe que l'expression renforcee de miR-181a dans les lymphocytes T matures provoque une augmentation marquee de l'activation des lymphocytes T et augmente la sensibilite des lymphocytes T aux antigenes peptidiques. En outre, les lymphoblastes T presentant une expression de miR-181a plus elevee deviennent reactifs aux antagonistes. Les effets de miR-181a sur la discrimination antigenique sont en partie obtenus par attenuation de l'expression de multiples regulateurs negatifs dans la voie de signalisation des recepteurs des lymphocytes T (TCR), y compris PTPN22 et les phosphatases a double specificite DUSP5 et DUSP6. Il en resulte une reduction du seuil de signalisation des TCR, d'ou un renforcement quantitatif et qualitatif de la sensibilite des lymphocytes T aux antigenes.

Jacqueline Chau

Papers

miR-181a is an intrinsic modulator of T cell sensitivity and selection

Identification of new microRNA genes and aberrant microRNA profiles in childhood acute lymphoblastic leukemia.

Modulation of T cell signaling threshold and T cell sensitivity to antigens

Modulation du seuil de signalisation des lymphocytes t et de la sensibilité des lymphocytes t aux antigènes

Modulation einer t-zellen-signalisierungsschwelle und der empfindlichkeit von t-zellen gegen antigene