The role of microRNAs in cell:cell communication

Lead Research Organisation: Queen's University of Belfast
Department Name: Medicine Dentistry and Biomedical Sci

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description For cells to function in the required fashion they must regulate the expression of thousands of genes appropriately. MicroRNAs are small RNA molecules which have recently been shown play a critical role in this process. It has been suggested that these microRNAs can be released from cells and regulate the expression of genes in target cells. During this project we studied both the microRNAs present within cells grown in the laboratory and those released from these cells in microscopic vesicles. We obtained millions of nucleotide sequences which identified all the different microRNAs present. The first key finding was that the profile of microRNAs in the cells was distinct from that present in the vesicles. This provided evidence to support the hypothesis that microRNAs are selectively exported from cells.

The second key finding was that it is possible to manipulate the microRNA content of the vesicles. When a specific microRNA was artificially over-expressed in cells, this microRNA was shown to subsequently appear in the vesicles. The over-expressed microRNA comprised a significant proportion of all microRNAs detected in the vesicles. The significance of this finding is that it demonstrates the potential of modifying vesicular microRNAs for future experimental and biomedical applications.

The third key finding was to confirm that vesicles released from one cell type could deliver microRNAs to another cell type and affect gene expression. The fusion of fluorescently labeled vesicles with endothelial cells was followed using confocal microscopy. The effects of microRNAs transferred in this manner upon endothelial gene expression were detected using techniques to assay both global gene expression and specific candidate genes in detail.
Exploitation Route I envisage the research outcomes being taken forward in the biomedical field. The demonstration that microRNAs are selectively exported from cells provides a mechanistic basis for the use of circulating microRNAs as an indicator of disease status. Indeed the potential of microRNAs as biomarkers is now being widely exploited both academically and commercially and is contributing to the development of personalised healthcare.
The demonstration that vesicular microRNAs can be modified and then delivered to a target cells supports their development as therapeutics
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The publications arising from this project have influenced the microRNA field, as evidenced by multiple citations (85 to date for the principal publication). Together with the findings of many related studies, this knowledge has advanced the development of the use circulaing microRNAs as biomarkers. This is beginning to have an impact upon health.
First Year Of Impact 2014
Sector Healthcare
Impact Types Societal

 
Description Micro-RT-PCR-Seq: An assay to detect and quantify novel patterns of microRNA expression
Amount £104,950 (GBP)
Funding ID POC 450 
Organisation Invest Northern Ireland 
Sector Public
Country United Kingdom
Start 12/2013 
End 11/2014
 
Description Proximity to Dicovery: Innovation and implementation of small RNA sequencing technologies with Bioo Scientific
Amount £25,401 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2017 
End 10/2017
 
Description Research Grant: Identification of microRNAs as early indicators of diabetic kidney disease
Amount £22,000 (GBP)
Organisation Kidney Research Fund Northern Ireland 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 12/2018
 
Description Vesicular delivery of microRNAs to enhance regeneration of ischaemic retina
Amount £169,540 (GBP)
Funding ID 1444/45 
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2013 
End 11/2016
 
Title Intracellular and extracellular microRNAs expressed by HEK293T cells 
Description MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. microRNAs can be detected using high throughput sequencing. This dataset includes the sequences generated from such an experiment conducted on HEK293T cells and the vesicles which they release, before or after transfection with a vector for miR-146 overexpression. 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? No  
Impact No actual impacts realised to date 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38916
 
Title Small RNA sequencing of plasma RNA 
Description High throughput sequencing (NGS) data from small RNA extracted from plasma samples 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact No notable impacts of which I am aware to date 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52981
 
Description Norther Ireland Science Festival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I organised a series of short talks highlighting the research within my Institute, the Centre for Experimental Medicine at Queen's University. These included a presentation about my own research. This was part of an event called 'Know Your Enemy', highlighting Disease-Focussed Research within the University on Saturday 17 February, 2018 which was itself part of the wider N. Ireland Science Festival.
The event was advertised as follows:
'Join scientists from the Centre for Experimental Medicine at Queen's to explore how the human body works and how we are leading the fight against disease. You will hear about the work of local scientists who are committed to improving the lives of patients, and who focus their efforts around three disease themes: eye disease, diabetes/vascular disease and respiratory/infectious disease. Come explore our exciting research and let us inspire you through a range of short talks, demonstrations and interactive stands, all led by the Centre's staff and students. The whole family can experience research up close and tours of our state-of- the-art building will allow visitors to appreciate our commitment to disease focused research'. Over 300 members of the public attended and feedback was overwhelmingly positive.
Year(s) Of Engagement Activity 2018
URL http://www.nisciencefestival.com/event.php?e=103