The role of microRNAs in cell:cell communication

The project investigates a novel form of communication between cells involving the transfer of small regulatory molecules called 'microRNAs' in small sacs or 'microvesicles'. Many cells generate microvesicles from their cell membranes, enclosing contents from inside the cell. The released microvesicles are carried in the blood, for example, until they contact another cell. They can then interact with the surface of the second cell or actually be taken up and release their contents inside. It has been shown that microvesicles convey 'instructions' telling the recipient cell to, for example, divide or form new blood vessels. Whilst microvesicles are ubiquitous in lower organisms, in vertebrates they have been isolated primarily from blood and other body fluids. It may be that routine passage of microvesicles from cell to cell within tissues has simply not been observed due to the difficulties of detection. If this is the case, they are a hugely underestimated form of communication. One of the classes of molecule carried by microvesicles is microRNA. Over the last few years these small RNAs have been shown to play an indispensable role in regulating gene expression and to be essential for normal development. Each of the ~500 microRNAs present in vertebrates targets several hundred messenger RNA molecules (mRNAs). MicroRNAs therefore have the capacity to 'fine tune' global gene expression patterns. The first objective of the project is to demonstrate that microRNAs carried by microvesicles can alter gene expression in their target cells. This has never been reported and would show that it is feasible for cells to communicate between one another using this mechanism. The second objective is to measure the extent and significance of this form of communication in biological systems. We have chosen to concentrate on a critical physiological process called angiogenesis, whereby new blood vessels are formed from pre-existing vessels. Angiogenesis relies on cell-cell communication and can be readily studied in the laboratory setting. A new technique which can simultaneously measure millions of RNA molecules will be used to investigate whether the microRNAs carried in microvesicles are affecting the expression of genes in the endothelial cells as they form new blood vessels. The main expected outcome of this project is knowledge of whether gene expression can be modulated by microRNAs carried by microvesicles and if this is an important mechanism. The several reports of microRNAs in microvesicles appear to represent a general phenomenon because we have replicated this finding in multiple cell types. Given the known and potentially widespread existence of microvesicles, microRNA transfer could be a significant novel form of inter-cellular communication. If this proves to be the case the beneficiaries of the project would be all those who are working in the field of cellular communication, for example during development. This knowledge could lead to better understanding of certain disease processes. This mechanism is amenable to manipulation and could therefore provide a novel approach for intervention in disease and industrial processes.

The aim of the proposed research is to test the hypothesis that 'MicroRNAs (miRNAs) mediate intercellular communication'. Small miRNA molecules regulate gene expression by binding to mRNA molecules with partially complementary target sites. Vertebrates have ~500 miRNAs, each of which can direct degradation or inhibit translation of many target genes. The regulatory potential of miRNAs is therefore immense. Whilst the role of miRNAs within their cell of origin is established they have recently been isolated from microvesicles released from a range of cells. The first main objective of the work is to show that miRNAs can be transferred between cells within microvesicles and be active in the target cell. A model system which is readily amenable to genetic manipulation will be employed: HEK293 cells will be transfected with a plasmid which directs overexpression of any chosen miRNA. Our pilot data suggest that microvesicles collected from the supernatant of such cultures are enriched for the overexpressed miRNA. These microvesicles will be incubated with fresh cells and the ability of the miRNA to be transferred tested using a fluorescent reporter system. The expression of predicted target genes of the overexpressed miRNA will be also be assessed. The ability of overexpressed miRNAs within microvesicles to modulate a critical physiological process will be assessed using an in vitro model of angiogenesis. The second main objective is to determine whether endogenous microvesicle-mediated miRNA signalling exerts a significant effect on angiogenic processes. Microvesicle signalling between endothelial progenitor cells and microvascular cells will be investigated. In order to study the global effects of miRNAs upon gene expression the most sensitive and quantitative method available, Next Generation Sequencing, will be employed. Shifts in the global pattern of target cell gene expression will be analysed to detect changes predicted to be directed by microvesicular miRNAs.

Who will benefit from this research? If the hypothesis that 'MicroRNAs mediate intercellular communication' is correct then there will be many beneficiaries. This will be particularly true if this form of communication is, as we suspect, more ubiquitous than currently appreciated. The immediate beneficiaries will be academic researchers working on RNA or in the cell signaling field. In the commercial private sector the first benefits are likely to be in the biotechnology or pharmaceutical industries. The use of microvesicles engineered to contain miRNAs that could regulate genes in, for example, a specific pathway, could have biotechnological applications. An understanding of this novel form of intercellular communication would both provide new targets for therapeutic intervention and provide a platform for the delivery of therapeutic agents. The use of RNAi to target specific genes is now widespread, however delivery remains an important issue and microvesicles have great potential in this regard. In the third sector images arising from the research could, for example, be used to promote research. Such images are already displayed within the Centre for Vision and Vascular Science and are appreciated by visitors for their intrinsic artistic value. How will they benefit from this research? The research has the potential to improve the nation's health if microvesicles are subsequently implicated in disease and therapies are developed to intervene to correct the dysfunction. Potential IPR stemming from the work will be channeled through the University's technology transfer program. Any commercialisation is likely to be pursued within the UK, which would increase wealth and foster economic competitiveness within a knowledge-based economy. It is difficult to predict a timescale, perhaps 10-20 years? If the hypothesis is supported then a follow-on study would have to be performed to confirm any potential biomedical significance. If this was positive, a further study would be required to develop a potential therapeutic agent which would then require extensive testing. What will be done to ensure that they benefit from this research? The results of the research will of course be published in open access peer-reviewed publications which are freely available for interested members of the public to read. However, to make the wider group of beneficiaries aware of the research and to make it available in a more accessible format the following steps will be taken. Firstly, a specific webpage will be created to communicate the ongoing research in a straightforward manner. Secondly, press releases summarising key findings will be prepared with the assistance of the University Marketing and Communications Office. Thirdly, the applicants will take all available opportunities to engage stakeholders through speaking at appropriate meetings and contributing to articles catering for audiences beyond the academic community.