Inhibition of long noncoding RNAs using LNA™ longRNA GapmeR LNA™ longRNA GapmeRs
Dr. Jean-Christophe Marine
Drs. Jean-Christophe Marine, Eleonora Leucci and Laura Standaert work at the Laboratory for Molecular Cancer Biology, K.U. Leuven in Belgium. They have been using LNA™ longRNA GapmeRs to silence non-coding RNAs linked to cancer.
What is the main focus of the research conducted in your lab?
Our research group focuses on the analysis of pathways that underlie the genesis, progression and maintenance of cancer. The goal is to understand how fundamental cellular processes in normal cells are controlled by the genes that are implicated in cancer. At the same time, we wish to understand the mechanisms by which mutations interfere with these natural processes to bring about tumor development and affect therapy outcome. We are currently studying the implication of a number of selected non-coding RNAs in p53 biology and/or melanoma progression.
How did you come to be interested in long noncoding RNAs?
There is an increased body of evidence indicating that lncRNAs play a key role in the etiology of several cancers. Through several profiling experiments we obtained evidence that a dozen lncRNAs are specifically expressed in metastatic melanoma. We now aim at exploring a putative causative link between the specific upregulation of these lncRNAs and melanoma development/progression. lncRNAs are indeed molecules that could be targeted in the context of targeted/combination therapy.
What is the aim of your current project?
Our aim is to efficiently and specifically silence the expression of melanoma-specific lncRNAs, which could be challenging due to the low expression level and their intracellular localization.
Which specific questions did you want to address?
We would like to know if melanoma cells rely on the expression of these lncRNAs for their proliferation and/or survival by deciphering the potential underlying molecular mechanism(s).
Which experiments had you performed leading up to this project?
We have assessed the expression levels of >1500 different lncRNAs by qPCR in a large panel of human cancer cell lines and identified a dozen lncRNAs of interest, which we will further investigate by evaluating the biological consequences of silencing these lncRNAs.
What, if any, was your previous experience with long noncoding RNA inhibition?
We have successfully used Exiqon’s LNA™ LongRNA GapmeRs to silence the highly abundant MALAT-1 in our previous testing of this new technology.
How did you perform the experiments and analyze the results?
With the assistance of Exiqon we had several gapmers designed for each lncRNA target including a scrambled control. For each lncRNA gapmer we assessed the target inhibition by qPCR to identify the most potent gapmer. We aimed at identifying at least two different gapmers for each target that lead to a decreased expression by 50% or more. Typically, we pre-screened about 10 different gapmers to identify two, of which we obtained silencing efficiency of more than 50% KD.
What do you find to be the main benefits of the LNA™ longRNA GapmeR from Exiqon?
LNA™ gapmers allow efficient targeting of lncRNAs which can be difficult to target by other technologies such as siRNA/shRNA. LNA™ gapmers are particularly suitable for silencing nuclear lncRNAs (which we have experienced difficulties to target by siRNAs).
What other products have you used, and how did they compare to Exiqon’s LNA™ longRNA GapmeR?
We have tried both siRNA and shRNA-based approaches to silence some of the lncRNAs. For example, these approaches were unsuccessful in knocking down an ultraconserved lncRNA that is expressed at a very low level, whereas efficient targeting was observed using LNA™ gapmers.
What would be your advice to colleagues about getting started with long noncoding RNA in vitro/in vivo inhibition?
When addressing a new target, we highly recommend performing a pre-screen with an optimized transfection setup for your working cell line.
In your opinion what is the most important factor for a successful long noncoding RNA inhibition experiment?
Unfortunately the KD efficiency is still relatively unpredictable as it is dependent on a numbers of parameters which are difficult to control such as expression levels, cellular localization etc. The pre-screen is therefore a necessity. It is also critical to use several controls such as scramble/non-targeting LNA™ gapmer as well as gapmers with several mismatches as negative controls. It is also important to show reproducibility –and dose dependency- of the biological effect with at least two different LNA™ gapmers targeting the lncRNA of interest.
What are the next steps in the current project and how do you plan to perform them?
Having successfully identified potent LNA™ gapmers for our selected lncRNAs of interest, we have obtained preliminary data showing important cellular phenotypes by inhibiting these lncRNAs. We are now confident in moving to the next step of confirming the biological consequences of this inhibition both in vitro
including analysis of the effect on cell proliferation, survival, migration, as well as the in vivo
effect on melanoma progression/metastasis. Our ultimate goal would be to develop lncRNA-based anti-melanoma therapeutics
When and where will we hear more about your studies?
We are in the process of collecting data for a peer reviewed publication