MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.icroRNAs (miRNAs) are a class of approximately 22 nucleotide noncoding RNAs that mediate posttranscriptional gene regulation by binding to and repressing specific messenger RNA targets. We and others previously demonstrated that miRNAs are present in the human circulation in a cell-free form and that altered plasma and serum miRNA profiles are observed in cancer and other diseases (1-9). This, along with the finding that miRNAs are remarkably stable in plasma despite high circulating RNase activity (1), suggests that miRNAs may be developed into a powerful new class of blood-based biomarkers.The mechanism underlying the unexpected stability of cell-free miRNAs in the RNase-rich environment of blood has not been systematically investigated, although it has important implications for miRNA biomarker development and for potential biological functions of circulating miRNAs (10). Currently, the dominant model for circulating miRNA stability is that miRNAs are released from cells in membrane-bound vesicles, which protect them from blood RNase activity. Vesicles proposed as carriers of circulating miRNAs include exosomes, which are 50-to 90-nm vesicles arising from multivesicular bodie...

Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.

Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.microvesicle | circulating

Epstein-Barr virus (EBV

Loss of the Epstein-Barr virus (EBV) genome from Akata Burkitt lymphoma (BL) cells is coincident with Epstein-Barr virus (EBV) is a ubiquitous human herpesvirusthat establishes a life-long latent infection within B lymphocytes. Latency is associated with the expression of as many as ten viral proteins, including a family of six nuclear proteins (EBNAs), three integral membrane proteins (LMPs) and the recently identified polypeptide 24). Several of these, most notably the EBNA-3 proteins, elicit a strong EBVspecific cytotoxic T-lymphocyte immune response (23,38,43). Through differential expression of EBV proteins that are immunogenic, potentially oncogenic and those necessary for sustaining infection, an equilibrium is established between infected B cells and the host's immune surveillance. As a consequence, a relatively stable pool of latently infected cells is maintained by the host (31,36,56). Thus, EBV is particularly well adapted for persistence within B lymphocytes of an immune host, and though the virus encodes the oncogenic protein LMP-1, its association with human malignancy is rare, especially considering the high incidence of EBV infection worldwide.As a result of the balanced relationship that exists between EBV and its host, pathogenesis associated with EBV latency is

The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.

The Epstein-Barr virus (EBV) EBER transcripts are small, highly structured RNAs able to bind to and inhibit activation of the double-stranded RNA-dependent protein kinase PKR in cell-free systems, and within latently infected B-cell lines they inhibit alpha interferon-induced apoptosis that is believed to be mediated through PKR. Here, we address the consequences of EBER expression for PKR activation in vivo in response to alpha interferon. In agreement with published findings, either EBV infection or the EBERs alone protected Burkitt lymphoma cells from alpha-interferon-induced apoptosis. However, utilizing multiple phosphorylation state-specific antibodies to monitor PKR activation within cells in response to interferon, we demonstrate that the EBERs are unable to inhibit phosphorylation of either cytoplasmic or nuclear PKR. Concordantly, a direct substrate of PKR, the ␣ subunit of eukaryotic initiation factor 2 (eIF-2␣), was equally phosphorylated in EBV-positive and EBV-negative cells following interferon treatment. Therefore, EBER inhibition of alphainterferon-induced apoptosis, and potentially other PKR-mediated events, is unlikely to be mediated through direct inhibition of PKR, as previously thought.

The Epstein-Barr virus (EBV)-encoded RNAs, EBER-1 and EBER-2, are highly abundant noncoding nuclear RNAs expressed during all forms of EBV latency. The EBERs have been shown to impart significant tumorigenic potential upon EBV-negative Burkitt lymphoma (BL) cells and to contribute to the growth potential of other B-cell lymphoma-, gastric carcinoma-, and nasopharyngeal carcinoma-derived cell lines. However, the mechanisms underlying this EBER-dependent enhancement of cell growth potential remain to be elucidated. Here we focused on the known interaction between EBER-1 and the cellular ribosomal protein L22 and the consequences of this interaction with respect to the growth-promoting properties of the EBERs. L22, a component of 60S ribosomal subunits, binds three sites on EBER-1, and a substantial fraction of available L22 is relocalized from nucleoli to the nucleoplasm in EBV-infected cells. To investigate the hypothesis that EBER-1-mediated relocalization of L22 in EBV-infected cells is critical for EBER-dependent functions, we investigated whether EBER-1 expression is necessary and sufficient for nucleoplasmic retention of L22. Following demonstration of this, we utilized RNA-protein binding assays and fluorescence localization studies to demonstrate that mutation of the L22 binding sites on EBER-1 prevents L22 binding and inhibits EBER-1-dependent L22 relocalization. Finally, the in vivo consequence of preventing L22 relocalization in EBERexpressing cells was examined in soft agar colony formation assays. We demonstrate that BL cells expressing mutated EBER-1 RNAs rendered incapable of binding L22 have significantly reduced capacity to enhance cell growth potential relative to BL cells expressing wild-type EBERs.