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Several studies have indicated an important role for miR-155 in the pathogenesis of B-cell lymphoma. Highly elevated levels of miR-155 were indeed observed in most B-cell lymphomas with the exception of Burkitt lymphoma (BL). However, the molecular mechanisms that underlie the oncogenic role of miR-155 in B-cell lymphoma are not well understood. To identify the miR-155 targets relevant for B-cell lymphoma, we performed RNA immunoprecipitation of Argonaute 2 in Hodgkin lymphoma (HL) cells upon miR-155 inhibition and in BL cells upon ectopic expression of miR-155. We identified 54 miR-155-specific target genes in BL cells and confirmed miR-155 targeting of DET1, NIAM, TRIM32, HOMEZ, PSIP1 and JARID2. Five of these targets are also regulated by endogenous miR-155 in HL cells. Both overexpression of miR-155 and inhibition of expression of the novel miR-155 target gene NIAM increased proliferation of BL cells. In primary B-cell lymphoma NIAM-positive cases have significant lower levels of miR-155 as compared to NIAM-negative cases, suggesting that NIAM is also regulated by miR-155 in primary B-cell lymphoma. Thus, our data indicate an oncogenic role for miR-155 in B-cell lymphoma which involves targeting the tumor suppressor NIAM.

T-cell activation affects microRNA (miRNA) expression in T-cell subsets. However, little is known about the kinetics of miRNA regulation and possible differences between CD4 and CD8 T cells. In this study we set out to analyze the kinetics of activation-induced expression regulation of twelve pre-selected miRNAs. The dynamics of the expression of these miRNAs was studied in sorted CD4 and CD8 CD45RO- T cells of healthy individuals stimulated with αCD3/αCD28 antibodies. Analysis of miRNA levels at day 3, 5, 7 and 10 showed significant activation-induced changes in expression levels of all twelve miRNAs. Expression levels of nine miRNAs, including miR-21, miR-146a and miR-155, were induced following activation, whereas expression of three miRNAs, including miR-31, were decreased following activation. The expression changes of miR-18a and miR-155 was relatively early, at day 3, whereas expression of miR-451, miR-21 and miR-146a was evident at day 5, 7 and 10, respectively. Four miRNAs showed a differential regulation between CD4 and CD8 T cells. Induction of miR-18a and miR-21 was more pronounced and occurred earlier in CD4 T cells compared to CD8 T cells. Downregulation of miR-223 and miR-451 was also more pronounced in CD4 T cells compared to CD8 T cells. In conclusion, we show a complex pattern of miRNA expression regulation upon T-cell activation with early and late as well as CD4 and CD8 T cell specific changes. These differences might be the result of differences in kinetics and efficiency of CD4 and CD8 T-cells in response to antigen priming.

Disturbed expression of microRNAs (miRNAs) in regulatory T cells (Tregs) leads to development of autoimmunity in experimental mouse models. However, the miRNA expression signature characterizing Tregs of autoimmune diseases, such as rheumatoid arthritis (RA) has not been determined yet. In this study, we have used a microarray approach to comprehensively analyze miRNA expression signatures of both naive Tregs (CD4+CD45RO-CD25++) and memory Tregs (CD4+CD45RO+CD25++), as well as conventional naive (CD4+CD45RO+CD25+) and memory (CD4+CD45RO+CD25+) T cells (Tconvs) derived from peripheral blood of RA patients and matched healthy controls. Differential expression of selected miRNAs was validated by TaqMan-based quantitative reverse transcription-PCR. We found a positive correlation between increased expression of miR-451 in T cells of RA patients and disease activity score (DAS28), erythrocyte sedimentation rate levels and serum levels of interleukin-6. Moreover, we found characteristic, disease-and treatment-independent, global miRNA expression signatures defining naive Tregs, memory Tregs, naive Tconvs and memory Tconvs. The analysis allowed us to define miRNAs characteristic for a general naive phenotype (for example, miR-92a) and a general memory phenotype (for example, miR-21, miR-155). Importantly, the analysis allowed us to define miRNAs that are specifically expressed in both naive and memory Tregs, defining as such miRNA signature characterizing the Treg phenotype (that is, miR-146a, miR-3162, miR-1202, miR-1246 and miR-4281).

MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We describe here a straightforward method to generate retroviral miRNA sponge constructs using a single directional ligation reaction. This approach allows generation of sponges containing more than 20 miRNA binding sites. We provide a basis for the design of the sponge constructs with respect to the sequence of the miRNA binding site and the sequences flanking the miRNA binding sites. In-silico validation approaches are presented to test the predicted efficiencies of the sponges in comparison to known target genes. In addition, we describe in vitro validation experiments to confirm the effectiveness of the miRNA sponges. Finally, we describe how the here described procedure can be adapted to easily generate sponges that target multiple miRNAs simultaneously. In summary, our approach allows rapid generation of single or combination miRNA sponges that can be used for long-term miRNA loss-of-function studies. (C) 2012 Elsevier Inc. All rights reserved.

Objective. The aim of this study was to investigate the turnover of Treg cells in the SF of RA patients.Methods. Treg cells were enumerated in peripheral blood and SF of RA patients and analysed by flow cytometry for expression of the proliferation marker Ki-67 and binding of the apoptosis marker annexin V. Sorted Treg cells of RA patients were analysed for expression of anti-apoptotic regulators Bcl-2 and microRNA-21 (miR-21) by RT-PCR.Results. Treg cells displaying a memory phenotype were abundant in the SF of RA patients. SF Treg cells more frequently expressed the proliferation marker Ki-67 than conventional T cells. Only few SF Treg cells were apoptotic, as indicated by limited annexin V staining of these cells. SF Treg cells displayed high transcription levels of Bcl-2 and miR-21 in comparison with SF conventional T cells and peripheral blood Treg cells.Conclusion. Treg cells with a memory phenotype accumulate in the SF of RA patients. These Treg cells have a high proliferative activity and demonstrate little apoptosis. The latter finding could be explained by high transcription of Bcl-2 and miR-21 in SF Treg cells.

MicroRNAs (miRNAs) are instrumental to many aspects of immunity, including various levels of T-cell immunity. Over the last decade, crucial immune functions were shown to be regulated by specific miRNAs. These immuno-miRs' regulate generic cell biological processes in T cells, such as proliferation and apoptosis, as well as a number of T-cell-specific features that are fundamental to the development, differentiation and function of T cells. In this review, we give an overview of the current literature with respect to the role of miRNAs at various stages of T-cell development, maturation, differentiation, activation and ageing. Little is known about the involvement of miRNAs in thymic T-cell development, although miR-181a and miR-150 have been implicated herein. In contrast, several broadly expressed miRNAs including miR-21, miR-155 and miR-17 similar to 92, have now been shown to regulate T-cell activation. Other miRNAs, including miR-146a, show a more T-cell-subset-specific expression pattern and are involved in the regulation of processes unique to that specific T-cell subset. Importantly, differences in the miRNA target gene repertoires of different T-cell subsets allow similar miRNAs to control different T-cell-subset-specific functions. Interestingly, several of the here described immuno-miRs have also been implicated in T-cell ageing and there are clear indications for causal involvement of miRNAs in immunosenescence. It is concluded that immuno-miRs have a dynamic regulatory role in many aspects of T-cell differentiation, activation, function and ageing. An important notion when studying miRNAs in relation to T-cell biology is that specific immuno-miRs may have quite unrelated functions in closely related T-cell subsets.

Immune cell-type specific miRNA expression patterns have been described but the detailed role of single miRNAs in the function of T-cells remains largely unknown. We investigated the role of miR-21 in the function of primary human CD4+ T-cells. MiR-21 is substantially expressed in T-cells with a memory phenotype, and is robustly upregulated upon alpha CD3/CD28 activation of both naive and memory T-cells. By inhibiting the endogenous miR-21 function in activated naive and memory T-cells, we showed that miR-21 regulates fundamentally different aspects of T-cell biology, depending on the differentiation status of the T-cell. Stable inhibition of miR-21 function in activated memory T-cells led to growth disadvantage and apoptosis, indicating that the survival of memory T-cells depends on miR-21 function. In contrast, stable inhibition of miR-21 function in activated naive T-cells did not result in growth disadvantage, but led to a significant induction of CCR7 protein expression. Direct interaction between CCR7 and miR-21 was confirmed in a dual luciferase reporter assay. Our data provide evidence for a dual role of miR-21 in CD4+ T cells; Regulation of T-cell survival is confined to activated memory T-cells, while modulation of potential homing properties, through downregulation of CCR7 protein expression, is observed in activated naive T-cells.