MicroRNA function in endothelial cellsSolving the mystery of an unknown target gene using microRNA Target Site Blockers
Dr. Virginie Mattot
Dr. Virgine Mattot works in the team “Angiogenesis, endothelium activation and Cancer” directed by Dr. Fabrice Soncin at the Institut de Biologie de Lille in France where she studies the roles played by microRNAs in endothelial cells during physiological and pathological processes such as angiogenesis or endothelium activation. She has been using Target Site Blockers to investigate the role of microRNAs on putative targets which functions are yet unknown.
What is the main focus of the research conducted in your lab?
We are studying endothelial cell functions with a particular interest in angiogenesis and endothelium activation during physiological and tumoral vascular development.
How did your research lead to the study of microRNAs?
A few years ago, we identified in my team a new endothelial cell-specific gene which harbors a microRNA in its intronic sequence. We have since been working on understanding the functions of both this new gene and its intronic microRNA in endothelial cells.
What is the aim of your current project?
While we were searching for the functions of the intronic microRNA, we identified an unknown gene as a putative target. The aim of my project was to investigate if this unknown gene was actually a genuine target and if regulation of this gene by the microRNA was involved in endothelial cell function. We had already characterized the endothelial cell phenotype associated with the inhibition of our intronic microRNA. We then used miRCURY LNA™ Target Site Blockers to demonstrate that the expression of this unknown gene is actually controlled by this microRNA. Further, we also demonstrated that the microRNA regulates specific endothelial cell properties through regulation of this unknown gene.
How did you perform the experiments and analyze the results?
LNA™ enhanced target site blockers (TSB) for our microRNA were designed by Exiqon. We transfected the TSBs into endothelial cells using our standard procedure and analysed the induced phenotype. As a control for these experiments, a mutated version of the TSB was designed by Exiqon and transfected into endothelial cells. We first verified that this TSB was functional by analyzing the expression of the miRNA target against which the TSB was directed in transfected cells. Finally, we showed that the TSB induced similar phenotypes as those found when we inhibited the microRNA in the same cells.
What were some specific challenges in your experiments and how did you overcome them?
The fact that the target gene for our microRNA was unknown was a major challenge. Without specific available tools, like antibodies, it becomes difficult to demonstrate the effect of the microRNA on the gene in question and to show that the unknown gene is indeed responsible for the functions of the microRNA. However through the use of specific target site blockers, we were able to demonstrate that this unknown gene was associated with the phenotype observed when the microRNA was inhibited in endothelial cells.
How do you feel about your results so far?
We are very pleased with the results of the TSB experiments and altogether these results demonstrate that our miRNA of interest is functional in endothelial cells through the regulation of a target gene with a previously unknown role.
What do you find to be the main benefits/advantage of the LNA™ microRNA target site blockers from Exiqon?
Target Site Blockers are efficient tools to demonstrate the specific involvement of putative microRNA targets in the function played by this microRNA. The use of LNA™ allows the design of short oligonucleotides that are very specific and easy to work with.
What would be your advice to colleagues about getting started with microRNA functional analysis?
In order to address the role played by a microRNA, it is essential to perform both gain and loss of functions experiments.
What are the next steps in the current project and how do you plan to perform them?
We plan to use microRNA inhibitor libraries to identify more microRNAs specifically involved in the processes that we currently study.
When and where will be hear /read more about your studies?
Hopefully really soon because we are currently in the process of submitting a manuscript regarding the function of my microRNA of interest.