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Application Stories

NGS identifies therapeutic microRNA targets for skeletal disorders

mRNA and microRNA NGS Services combined with In vivo miRCURY LNA™ microRNA inhibitor studies

Dr. Hanna Taipaleenmäki

Dr. Hanna Taipaleenmäki is a research scientist within the group of Prof. Dr. Dr. Eric Hesse, in the Molecular Skeletal Biology Laboratory at the University Medical Center Hamburg-Eppendorf in Germany.

What is the main focus of your research?

The lab’s main research interest is bone biology. We are interested in several skeletal disorders – mainly osteoporosis and breast cancer bone metastases. Our goal is to better understand the pathological mechanisms contributing to these diseases and to develop novel therapeutic approaches.

What is the specific aim of the current project?

The main purpose of this study was to identify microRNAs that are regulated in response to bone anabolic treatments (therapies that promote bone formation). We thought that if we can then modulate the expression of these microRNAs, we may be able to recapitulate the bone anabolic effect of the treatment. Therefore these microRNAs may be potential new targets for novel bone anabolic therapies to treat skeletal disorders.

We used a well-established mouse model and applied two different bone anabolic treatments –one that is already approved for use in humans and a novel upcoming drug. We then harvested the bones without the bone marrow, extracted total RNA and sent it to Exiqon Services to perform microRNA and RNA sequencing from the exact same RNA samples.

“Exiqon Services performed microRNA and RNA sequencing from the exact same RNA samples.”

We were able to identify microRNAs that are downregulated in response to the bone anabolic treatments, and we have promising results indicating that inhibiting these microRNAs using In vivo LNA™ microRNA inhibitors might indeed be bone anabolic.

What, if any, was your previous experience with Next Generation Sequencing (NGS)?

We have done NGS before, but in collaboration with another group who had experience with NGS. This was our first time doing NGS ourselves.

What was the reason for choosing to perform the profiling using NGS?

We have done microRNA Arrays before, but now we really wanted to have a genome-wide analysis which provides much more information. In the future we also plan to look more into novel microRNAs, mRNAs and maybe even long non-coding RNAs.

Why did you decide to sequence both microRNA and mRNA?

Sequencing both microRNA and mRNA enabled us to identify changes on the microRNA level, and also to investigate what is happening to gene expression at the mRNA level, to understand which pathways are activated or downregulated as a result of the bone anabolic treatments. We were able to combine the microRNA and mRNA sequencing results to identify potential mRNA targets of specific microRNAs, and pathways that are regulated by these microRNAs.

Were there any challenges during the NGS project and how did you overcome them?

Bone tissue itself was the main challenge. Over the years we have established good protocols for RNA isolation from bone in our laboratory but even so it was challenging to get a good amount of RNA of sufficient quality for sequencing both microRNA and mRNA.

The RNA quality was at the lower end of the acceptable range, and we trusted Exiqon’s advice that it was good enough to proceed with sequencing. With the sequencing itself there were absolutely no problems - we were able to obtain sufficient reads from our samples, and the RNA quality did not appear to be a problem.

Did you identify any interesting candidates from your NGS data?

From the NGS experiments we identified some microRNA candidates whose expression levels are downregulated in response to both of the two bone anabolic treatments. Some of the microRNAs we identified by NGS have been reported before in bone, but many have not. This is most likely because there are very few microRNAs that have been identified as being expressed in bone in vivo – very few sequencing studies have been done so far on bone due to the technical challenges.

How did you investigate the potential therapeutic applications of the candidates you identified by NGS?

We thought that if we can then inhibit these candidate microRNAs, we may be able to recapitulate the bone anabolic effect of the treatment, and this might offer a potential approach to develop new therapies for skeletal disorders.

So, we got some LNA™ microRNA inhibitors from Exiqon and we have very promising results indicating that inhibition of those microRNAs might indeed be bone anabolic. We first characterized some microRNA candidates with in vitro functional studies, and now we are finishing the in vivo studies using the LNA™ microRNA inhibitors in mouse models. We found that inhibition of even a single microRNA has a bone anabolic effect. We have not done any synergistic studies yet.

It is particularly exciting that we have been able to deliver the In vivo LNA™ microRNA inhibitors into the bone. We also delivered In vivo FAM-labelled LNA™ microRNA inhibitors intravenously to see where they go, and we were really positively surprised to see high signals in bone – that’s pretty novel.

“We have been combining the NGS results with functional studies using [In vivo] LNA™ microRNA inhibitors in mouse models.”

What were important aspects for you when choosing an RNA service partner?

Reliability is absolutely important for this type of analysis, so that you know that things are done correctly. The people – it’s important to get support when needed and good communication. Time and cost are also important. It doesn’t have to be the cheapest but you have to get what you want for the money.

Why did you choose Exiqon as your service partner?

We were discussing with another company but we have had such a good experience with Exiqon with everything over the years that we decided to choose Exiqon. Another important factor was that Exiqon provides analysis of the sequencing data, which was very important for us.

“Exiqon provides analysis of the sequencing data, which was very important for us.”

What would be your advice to colleagues about getting started with RNA NGS today?

The important things are to select the service provider carefully since it is a very delicate procedure. Also bear in mind that you get a LOT of data. If you don’t have any experience in bioinformatics analysis, and you don’t have a provider who can offer you support or data analysis, then you might be lost.

“Select your service provider carefully since [RNA-Seq] is a very delicate procedure.”

In this respect it is good that Exiqon processes the data, and you get the raw data, but you also get a further analysis of the data. Also, because there is such a vast amount of data – think very carefully about the questions you want to ask and what you want to get out of the experiment, and perform the analysis based on these specific questions (or ask your provider to).

“With NGS you get a LOT of data…if you don’t have experience in bioinformatics you might be lost.”

How did you go about doing a combined analysis of the microRNA and mRNA sequencing data?

The data analysis provided by Exiqon was very good. Once we had identified the candidate microRNAs we then did a more manual analysis to identify mRNAs predicted to be regulated by those microRNAs (we used miRWalk or TargetScan), and look at the pathways. We also looked at the predicted and validated mRNA targets provided by Exiqon’s miRSearch analysis, and there were some overlapping targets.

What are the next steps in the current project and how do you plan to perform them?

First of all we are going to characterize the in vivo potential therapeutic effect of the candidate microRNAs in detail. To identify the potential mechanism of action for these microRNAs, we will use the mRNA sequencing data. Then we will look in even more detail at both the microRNA and mRNA sequencing datasets to see if there are any regulated microRNA or mRNAs, which are completely novel for bone. It’s a lot of data so I think there will be a lot of work for the future!

What are the future perspectives for this research?

There are challenges in developing microRNA inhibitor-based drugs (tissue specificity, targeted delivery, etc.) but I do think that microRNAs may have a place in the clinic in the future.

When and where will we hear /read more about your studies?

We hope to report our findings within the next year.

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