Viral miRNA delivered by exosomes from Marek's disease virus-transformed lymphoma cell line exerts regulatory function in internalized primary chicken embryo fibroblast cells.

In the past decade, research has demonstrated that viral miRNAs encoded by a number of viral genomes, particularly by most of the herpesvirus including Marek's disease virus (MDV), play important regulatory roles in viral infection, replication, and regulation of tumorigenesis. As macrovesicles in cells, exosomes can deliver viral miRNAs and exert gene regulatory functions. Whether the exosomes play a role in the replication, pathogenesis/tumorigenesis of avian herpesviruses such as oncogenic Marek's disease virus (MDV) remains unclear. Herein we extracted and identified the exosomes from MDV-transformed T cell line MSB-1 and demonstrated high abundance of MDV-1 miRNA expression. Using dual luciferase-based reporter assay, we also demonstrated that the exosomes derived from MSB-1 can deliver functional miRNA successfully into primary chicken embryo fibroblasts. These findings provide new insights into the role of exosomes and the mechanisms of how virus-encoded miRNA function in MDV latency/activation switching, viral replication, pathogenesis and/or tumorigenesis.

An unusual case of Brodie's abscess in the humerus of an adult female.

Brodie's abscess is a manifestation of subacute to chronic osteomyelitis, characterized as intraosseous abscess formation, usually on the metaphysis of the long tubular bones in the lower extremities of male pediatric patients. Clinically, Brodie's abscess presents with atraumatic bone pain of an insidious onset, with absence of systemic findings. Delay in diagnosis is common, as diagnostic imaging, followed by biopsy for culture and histologic examination are generally required to secure a diagnosis of Brodie's abscess. Treatment of Brodie's abscess is non-standardized, and usually consists of surgical debridement and antibacterial therapy. Despite the variability in therapeutic approaches, outcomes of Brodie's abscess treated with surgery and antibiotics are favourable. Herein we report a case of a delayed diagnosis of Brodie's abscess in the upper extremity of an adult female. While she improved with treatment of Brodie's abscess, the case serves to remind clinicians to consider this entity in adult individuals who present with atraumatic bone pain.

Monitoring the electroactive cargo of extracellular vesicles can differentiate various cancer cell lines.

Extracellular vesicles (EVs) are pivotal in cell-to-cell communication due to the array of cargo contained within these vesicles. EVs are considered important biomarkers for identification of disease, however most measurement approaches have focused on monitoring specific surface macromolecular targets. Our study focuses on exploring the electroactive component present within cargo from EVs obtained from various cancer and non-cancer cell lines using a disk carbon fiber microelectrode. Variations in the presence of oxidizable components were observed when the total cargo from EVs were measured, with the highest current detected in EVs from MCF7 cells. There were differences observed in the types of oxidizable species present within EVs from MCF7 and A549 cells. Single entity measurements showed clear spikes due to the detection of oxidizable cargo within EVs from MCF7 and A549 cells. These studies highlight the promise of monitoring EVs through the presence of varying electroactive components within the cargo and can drive a wave of new strategies towards specific detection of EVs for diagnosis and prognosis of various diseases.

Utilising extracellular vesicles for early cancer diagnostics: benefits, challenges and recommendations for the future.

To increase cancer patient survival and wellbeing, diagnostic assays need to be able to detect cases earlier, be applied more frequently, and preferably before symptoms develop. The expansion of blood biopsy technologies such as detection of circulating tumour cells and cell-free DNA has shown clinical promise for this. Extracellular vesicles released into the blood from tumour cells may offer a snapshot of the whole of the tumour. They represent a stable and multifaceted complex of a number of different types of molecules including DNA, RNA and protein. These represent biomarker targets that can be collected and analysed from blood samples, offering great potential for early diagnosis. In this review we discuss the benefits and challenges of the use of extracellular vesicles in this context and provide recommendations on where this developing field should focus their efforts to bring future success.

Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods.

Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

Detecting ovarian cancer using extracellular vesicles: progress and possibilities.

Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumor growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so, they represent an excellent potential source of novel biomarkers for the disease. In this review, we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but many studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insights into the stage of the disease and predict the likely therapeutic outcome. There remain many challenges to the use of EVs as biomarkers, but, through ongoing research and innovation in this exciting field, there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome.

Cisplatin induces the release of extracellular vesicles from ovarian cancer cells that can induce invasiveness and drug resistance in bystander cells.

Ovarian cancer has a poor overall survival that is partly caused by resistance to drugs such as cisplatin. Resistance can be acquired as a result of changes to the tumour or due to altered interactions within the tumour microenvironment. Extracellular vesicles (EVs), small lipid-bound vesicles that are loaded with macromolecular cargo and released by cells, are emerging as mediators of communication in the tumour microenvironment. We previously showed that EVs mediate the bystander effect, a phenomenon in which stressed cells can communicate with neighbouring naive cells leading to various effects including DNA damage; however, the role of EVs released following cisplatin treatment has not been tested. Here we show that treatment of cells with cisplatin led to the release of EVs that could induce invasion and increased resistance when taken up by bystander cells. This coincided with changes in p38 and JNK signalling, suggesting that these pathways may be involved in mediating the effects. We also show that EV uptake inhibitors could prevent this EV-mediated adaptive response and thus sensitize cells in vitro to the effects of cisplatin. Our results suggest that preventing pro-tumourigenic EV cross-talk during chemotherapy is a potential therapeutic target for improving outcome in ovarian cancer patients.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.

Mechanisms of Drug Resistance in Cancer: The Role of Extracellular Vesicles.

Drug resistance remains a major barrier to the successful treatment of cancer. The mechanisms by which therapeutic resistance arises are multifactorial. Recent evidence has shown that extracellular vesicles (EVs) play a role in mediating drug resistance. EVs are small vesicles carrying a variety of macromolecular cargo released by cells into the extracellular space and can be taken up into recipient cells, resulting in transfer of cellular material. EVs can mediate drug resistance by several mechanisms. They can serve as a pathway for sequestration of cytotoxic drugs, reducing the effective concentration at target sites. They can act as decoys carrying membrane proteins and capturing monoclonal antibodies intended to target receptors at the cell surface. EVs from resistant tumor cells can deliver mRNA, miRNA, long noncoding RNA, and protein inducing resistance in sensitive cells. This provides a new model for how resistance that arises can then spread through a heterogeneous tumor. EVs also mediate cross-talk between cancer cells and stromal cells in the tumor microenvironment, leading to tumor progression and acquisition of therapeutic resistance. In this review, we will describe what is known about how EVs can induce drug resistance, and discuss the ways in which EVs could be used as therapeutic targets or diagnostic markers for managing cancer treatment. While further characterization of the vesiculome and the mechanisms of EV function are still required, EVs offer an exciting opportunity in the fight against cancer.

Extracellular vesicles released following heat stress induce bystander effect in unstressed populations.

Cells naïve to stress can display the effects of stress, such as DNA damage and apoptosis, when they are exposed to signals from stressed cells; this phenomenon is known as the bystander effect. We previously showed that bystander effect induced by ionising radiation are mediated by extracellular vesicles (EVs). Bystander effect can also be induced by other types of stress, including heat shock, but it is unclear whether EVs are involved. Here we show that EVs released from heat shocked cells are also able to induce bystander damage in unstressed populations. Naïve cells treated with media conditioned by heat shocked cells showed higher levels of DNA damage and apoptosis than cells treated with media from control cells. Treating naïve cells with EVs derived from media conditioned by heat shocked cells also induced a bystander effect when compared to control, with DNA damage and apoptosis increasing whilst the level of cell viability was reduced. We demonstrate that treatment of naïve cells with heat shocked cell-derived EVs leads to greater invasiveness in a trans-well Matrigel assay. Finally, we show that naïve cells treated with EVs from heat-shocked cells are more likely to survive a subsequent heat shock, suggesting that these EVs mediate an adaptive response. We propose that EVs released following stress mediate an intercellular response that leads to apparent stress in neighbouring cells but also greater robustness in the face of a subsequent insult.

The Diagnostic and Prognostic Potential of microRNAs in Epithelial Ovarian Carcinoma.

Ovarian cancer causes more than 100,000 deaths globally per year. Despite intensive research efforts, there has been little improvement in the overall survival of patients over the past three decades. Most patients are not diagnosed until the cancer is at an advanced stage, by which time their chances of still being alive after 5 years are appallingly low. Attempts to extend life in these patients have been, for the most part, unsuccessful. This owes partly to the lack of suitable biomarkers for stratifying patients at the molecular level, into responders and non-responders. This would lead to more drugs being shown to have a clinical benefit and being approved for use in subgroups of patients. There is also a desperate need for improved biomarkers for earlier detection of ovarian cancer; if the disease is detected sooner there is a significantly improved outlook. In this review, we outline the evidence that microRNAs are deregulated in ovarian cancer, what this can tell us about tumour progression and how it could be used to improve patient stratification in clinical trials. We also describe the potential for circulating microRNAs, both associated with proteins or carried in vesicles, to be used as diagnostics for earlier detection or as biomarkers for informing clinicians on the prognosis and best treatment of ovarian cancer.

miRNAs and ovarian cancer: a miRiad of mechanisms to induce cisplatin drug resistance.

Ovarian cancer is the most aggressive gynecological cancer. One reason for the low 5-year survival rate of under 40% is that ovarian tumors usually acquire resistance to the platinum-based compounds used to treat them. Resistance to one such compound, cisplatin, can arise via numerous mechanisms that can be categorized as pre-, post-, on- or off-target. Pre-target mechanisms prevent accumulation of cisplatin in the cell, on-target mechanisms allow DNA damage to be repaired more efficiently, post-target mechanisms prevent the damage from inducing apoptosis and off-target mechanisms increase resistance via unrelated compensatory mechanisms. miRNAs are short non-coding RNAs that influence cellular function by repressing gene expression. Here we describe how miRNAs can induce cisplatin resistance in ovarian cancer cells via pre-, post-, on- and off-target mechanisms. A better understanding of how miRNAs feed into the mechanisms of drug resistance will inform the rational design of combination therapies for ovarian cancer.

Over-expression of miR-31 or loss of KCNMA1 leads to increased cisplatin resistance in ovarian cancer cells.

Ovarian cancers have a high mortality rate; this is in part due to resistance to the platinum-based compounds used in chemotherapy. In this paper, we assess the role of microRNA-31 in the development of chemoresistance to cisplatin. We used previous data from microarray experiments to identify potential microRNAs (miRNAs) involved in chemoresistance. The functional significance of these microRNAs was tested using miRNA mimics. We used RNA-seq to identify pathways and genes de-regulated in the resistant cell line and then determined their role using RNAi. Analysis of publically available datasets reveals the potential clinical significance. Our data show that miR-31 is increased, whilst potassium channel calcium activated large conductance subfamily M alpha, member 1 (KCNMA1), a subunit of calcium-regulated big potassium (BK) channels, is reduced in resistant ovarian cells. Over-expression of miR-31 increased resistance, as did knockdown of KCNMA1 or inhibition of BK channels. This suggests that these genes directly modulate cisplatin response. Our data also suggest that miR-31 represses KCNMA1 expression. Comparing the levels of miR-31 and KCNMA1 to cisplatin resistance in the NCI60 panel or chemoresistance in cohorts of ovarian cancer tumours reveals correlations that support a role for these genes in vitro and in vivo. Here we show that miR-31 and KCNMA1 are involved in mediating cisplatin resistance in ovarian cancer. Our data gives a new insight into the potential mechanisms to therapeutically target in cisplatin resistance common to ovarian cancer.

RNAi2015 - Ten years of RNAi Oxford.

The tenth RNAi conference was held at St. Hilda's College Oxford on the 24-26 March 2015. The conference offered researchers from all over the world the chance to present, discuss and discover work pertaining to the field of RNAi. RNAi has become an essential technique in genomic research for functional validation as well as an exciting avenue to explore in therapeutic medicine. Emerging techniques such as CRISPR as well as improvements in efficiency of existing techniques and expansions in libraries have cemented the importance of RNAi at the cutting edge of research. Featured presentations and posters showcased recent research in the field ranging from RNA detection in bio fluids through to potential oligonucleotide therapies.

The passenger strand, miR-21-3p, plays a role in mediating cisplatin resistance in ovarian cancer cells.

OBJECTIVE: Ovarian cancer is the deadliest gynaecological cancer. A major contributor to the poor survival rate is the development of chemoresistance to platinum-based therapies such as cisplatin and carboplatin. Here we aimed to test the role of miRNAs in the acquisition of drug resistance in ovarian cancer. METHODS: We used microarrays to measure miRNA levels in the ovarian cancer cell line A2780 and its cisplatin-resistant derivative CP70. The role of miRNAs and the mRNA targets were tested using transfected miRNA mimics and siRNAs, respectively. Potential in vivo significance was investigated by analysing RNA levels in cohorts of ovarian cancer patients. RESULTS: We identified several miRNAs that are increased in cisplatin-resistant cells. We show that most of these do not directly contribute to cisplatin resistance. Interestingly, miR-21-3p, the passenger strand of the known oncomiR, directed increased resistance to cisplatin in a range of ovarian cell lines. This effect was specific to the star strand, as miR-21-5p had the opposite effect and actually increased sensitivity of A2780 cells to cisplatin. We identify NAV3 as a potential target of miR-21-3p and show that knockdown of NAV3 increases resistance. Exosomes released by CP70 cells were also capable of increasing resistance in A2780 cells, although this was independent of miR-21-3p. Finally, we use publically available transcriptomic data to demonstrate that miR-21-3p is raised, while NAV3 is reduced, in ovarian tumours that are resistant to platinum treatment. CONCLUSION: Our data suggest that miR-21-3p can induce cisplatin resistance in ovarian tumours, potentially by targeting the NAV3 gene.

Meta-analysis using a novel database, miRStress, reveals miRNAs that are frequently associated with the radiation and hypoxia stress-responses.

Organisms are often exposed to environmental pressures that affect homeostasis, so it is important to understand the biological basis of stress-response. Various biological mechanisms have evolved to help cells cope with potentially cytotoxic changes in their environment. miRNAs are small non-coding RNAs which are able to regulate mRNA stability. It has been suggested that miRNAs may tip the balance between continued cytorepair and induction of apoptosis in response to stress. There is a wealth of data in the literature showing the effect of environmental stress on miRNAs, but it is scattered in a large number of disparate publications. Meta-analyses of this data would produce added insight into the molecular mechanisms of stress-response. To facilitate this we created and manually curated the miRStress database, which describes the changes in miRNA levels following an array of stress types in eukaryotic cells. Here we describe this database and validate the miRStress tool for analysing miRNAs that are regulated by stress. To validate the database we performed a cross-species analysis to identify miRNAs that respond to radiation. The analysis tool confirms miR-21 and miR-34a as frequently deregulated in response to radiation, but also identifies novel candidates as potentially important players in this stress response, including miR-15b, miR-19b, and miR-106a. Similarly, we used the miRStress tool to analyse hypoxia-responsive miRNAs. The most frequently deregulated miRNAs were miR-210 and miR-21, as expected. Several other miRNAs were also found to be associated with hypoxia, including miR-181b, miR-26a/b, miR-106a, miR-213 and miR-192. Therefore the miRStress tool has identified miRNAs with hitherto unknown or under-appreciated roles in the response to specific stress types. The miRStress tool, which can be used to uncover new insight into the biological roles of miRNAs, and also has the potential to unearth potential biomarkers for therapeutic response, is freely available at http://mudshark.brookes.ac.uk/MirStress.