Long non-coding RNAs in the cardiovascular system lncRNA silencing using LNA™ GapmeRs
Katharina Michalik, Reinier Boon
Dr. Stefanie Dimmeler
Dr. Reinier Boon
Katharina M. Michalik
and Stefanie Dimmeler
work at the Institute for Cardiovascular Regeneration at the Goethe University in Frankfurt, Germany. They are interested in microRNAs and long non-coding RNAs (lncRNAs) that control cardiovascular functions in endothelial cells.
What is the background for your current project involving LNA™ GapmeRs?
Endothelial cells control pivotal functions of the cardiovascular system. We hypothesized that lncRNAs are important for endothelial cell function and performed next generation sequencing experiments with human endothelial cells to identify lncRNAs that are present in endothelial cells.
What is the specific aim of your current project?
Our broad aim is to identify the role of lncRNAs in endothelium. Specifically, we wanted to address whether MALAT1, which is highly expressed in endothelial cells, is involved endothelial cell function.
What, if any, was your previous experience with gene silencing?
We use siRNA–mediated gene silencing as a standard tool in the lab and also have quite some experience with antimirs. siRNAs also worked to silence MALAT1, but LNA™ GapmeRs are more efficient. Next, we used LNA™ GapmeRs to silence Malat1 in mice in vivo
, which worked well.
How did you perform the experiments and analyze the results?
We transfect LNA™ GapmeRs with Lipofectamine RNAiMax (Life Technologies) for 48h and check the knockdown using qPCR. For the in vivo
experiments we inject intraperitoneally 20mg/kg of LNA™ GapmeRs.
We observed that MALAT1 expression was very strongly induced by hypoxia. This lead us to study the role of MALAT1 in angiogenesis. We used both siRNA and gapmers for MALAT1 knockdown and critically they gave very similar phenotypes. We used in vitro
spheroid angiogenesis and scratched wound assays. Silencing of MALAT1 increased VEGF independent basal endothelial cell migration and sprouting and disturbed sprouting in the presence of VEGF. We also investigated the effect on gene expression with microarrays and observed significant changes in expression of genes involved in cell cycle and DNA replication. Consistent with these data we found that MALAT1 KD was accompanied by reduced proliferation. In vivo
we observed a reduction in vessel density in the retina of neonatal MALAT1-/- knock-out mice. Using LNA gapmers we were able to further validate that MALAT1 is required for neovascularization this time in postnatal mice. We used a hind limb ischemia mouse model. With the gapmers we achieved efficient MALAT1 knockdown in the calf muscle which significantly reduced blood flow recovery and capillary density 21 days after induction of hind limb ischemia. Again we were able to use two different approaches to corroborate our findings – transgenic knockout mice and KD achieved by systemic administration of gapmers.
What were some specific challenges in your experiments?
Human MALAT1 and mouse Malat1 are quite homologues in comparison to other lncRNAs, but the sequences still differ quite a lot, which means that GapmeRs designed against human MALAT1 do not necessarily inhibit mouse Malat1.
How did you overcome them?
We were lucky that the LNA™ GapmeR that worked best for mouse Malat1 only had one mismatch with human MALAT1 and also silenced human MALAT1 efficiently.
How do you feel about your results so far?
We are very pleased with our results, which show for the first time that the lncRNA MALAT1 controls endothelial cell function.
What do you find to be the main benefits of the LNA™ GapmeRs from Exiqon?
Knock down works efficiently in vitro
and in vivo
. It is currently the best tool to knock down lncRNAs in vivo
What would be your advice to colleagues about getting started with gene silencing/inhibition experiments?
If possible find a LNA™ GapmeR that works for human and mouse. Test more than two LNA™ GapmeRs and test them in different concentrations.
In your opinion what is the most important factor for a successful gene silencing experiment?
We have tested about 10 GapmeRs against Malat1 and 7 of the sequences worked very efficiently in vitro
. We did not search for reasons that underlie the lack of knock down efficiency of the 3 GapmeRs that did not work. Possible reasons might be the secondary structure of the lncRNA or motives that are bound by RNA binding proteins that may limits accessibility.
What are the future perspectives for this research?
Identification of lncRNAs that may be targeted for development of therapeutic strategies against cardiovascular diseases.
When and where will be hear /read more about your studies?
Our study is just published online and will follow soon in print: Michalik et al. Circ Res. 2014.