The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of nuclear factor B (NF-B). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative reverse transcription-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-B binding sites in the miR-146a promoter and identified a role for an Oct-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a-expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon-responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.

The cellular microRNA miR-155 has been shown to be involved in lymphocyte activation and is expressed in

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We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF.hsa-mir199a ͉ hsa-mir346 ͉ stem cells ͉ plasticity H uman multipotent stromal cells (hMSCs) from bone marrow, also known as mesenchymal stem cells, are progenitor cells capable of differentiating into a variety of mature tissues (1). hMSCs can be isolated easily as single cell clones and differentiated into osteoblasts and adipocytes as well as other cellular phenotypes in culture. The cells hold great promise for therapy but the molecular mechanisms that govern the plasticity and differentiation remain unclear. Recently, the existence and function of a class of small, noncoding RNA molecules known as microRNAs (miRNAs) have gained attention as regulatory molecules. These genomically encoded RNAs undergo several modifications before being converted into mature 21-23 base pair transcripts capable of gene silencing. Biogenesis of functional miRNAs involves several enzymes, including Dicer and Drosha. Previous studies demonstrated that decreased expression of Dicer in Drosophila, zebrafish, and mice restricts the differentiation potential of stem cells (2-5). Studies have also demonstrated that specific miRNAs regulate gene expression during germ line development and cellular differentiation (2-6), including playing key roles in hematopoiesis as well as myogenic and neurogenic function (7-11). However, much of the work on miRNAs has focused on simpler organisms with very little data on human miRNAs. In this study we investigated the role of miRNAs in hMSCs, focusing first on the need for miRNA processing enzymes in hMSC differentiation. Then, we identified the key miRNAs that may regulate differentiation of hMSCs. Finally, we demonstrated that two of the miRNAs target leukemia inhibitory factor (LIF), the expression of which decreases as hMSCs differentiate. Results Generation of a Stable Knockdown of Dicer and Drosha in hMSCs.As a first step toward identifying the role of miRNAs in the differentiation of hMSCs, we analyzed the effect of decreasing the expression of key proteins...

The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lower in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.