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microRNAs in soft tissue sarcoma

miRCURY LNA™ microRNA Array
 
Caroline Gits
Caroline Gits
Caroline M.M. Gits works at the Department of Medical Oncology, Erasmus MC Cancer Institute in The Netherlands. She has been using Exiqon’s miRCURY LNA™ microRNA Arrays to characterize tumor-specific microRNAs.

What is the main focus of your research?

My PhD-project was concerned with the molecular characterization of soft tissue sarcomas, a group of rare, heterogeneous tumors of mesenchymal origin. The research mainly focused on the microRNA profiling in diverse (sub)types of soft tissue sarcomas, and the functional characterization of tumor specific microRNAs, thereby focusing on relevant microRNA target genes.


Why is this an important project?

Because of the rarity, the large number of entities, and the heterogeneity of soft tissue sarcomas, histological classification and prediction of clinical behavior and prognosis in sarcomas is often a challenge. Furthermore, there are few effective therapies for advanced soft tissue sarcomas. Because of the specific genetic abnormalities that some sarcoma subtypes exhibit, we would like to exploit these defects as therapeutic targets. A better understanding of the biology of soft tissue sarcoma subtypes, especially regarding driver genes and microRNAs that are involved in the tumorigenic process, could not only be useful for sarcoma (subtype) classification, but could also aid the development of new targeted therapies.


What was the reason for choosing to perform microRNA profiling using microarrays?

Microarray technology is relatively cheap and easy to use and analyze. We use total RNA (which we can also use for other applications), which is fluorescently labeled and hybridized to the microRNA capture probes on the microarrays. There is no need for isolation of the microRNA fraction or pre-amplification steps.


What was the specific aim of the microRNA profiling studies?

The aim was to identify soft tissue sarcoma (subtype) specific microRNAs. Subsequently we wanted to identify microRNA target genes and determine the function of these microRNAs in the tumors. We wanted to address the following questions:
1. Which microRNA are specifically deregulated in sarcoma (sub)types and can they be used to define diagnostic signatures?
2. Which of the deregulated tumor microRNAs play an essential role in the tumorigenic process?
3. Can specific microRNAs be used for therapeutic purposes?


How did you perform the experiments and analyze the results?

Total RNA was fluorescently labeled with Cy3, and subsequently hybridized with locked nucleic acid (LNA™) modified oligonucleotide capture probes (Exiqon) spotted in duplicate on Nexterion E slides. Hybridized slides were scanned and median spot intensity was determined. After background subtraction, expression values were quantile normalized, bad spots were deleted, and duplicate spots were averaged. For each expression value, the ratio to the geometric mean of the microRNA was log2 transformed. These values were used to determine differentially expressed microRNAs.


What were some specific challenges in your experiments?

The LNA™ capture probes were spotted in-house on Nexterion E slides. We did observe some small differences in spot intensity between the different print batches.


How did you overcome them?

For every experiment we had to use microarrays from a single print batch whenever possible. When comparing results from microarrays from different print batches an additional step of data normalization was applied.


How do you feel about your results from the microRNA array profiling experiments?

We feel very confident about the results. Even small differences in microRNA expression were consistently observed in samples from the same tumor (sub)type. In addition, microRNA families and microRNA cluster members were frequently found to be co-regulated. In most cases our microarray results could be validated by qPCR analyses.


What do you find to be the main benefits of the Exiqon LNA™ microRNA arrays?

The Exiqon LNA™ microRNA arrays enable profiling of many microRNAs in a large number of samples in a relatively short time span, with uncomplicated analyses, giving reliable and reproducible results.


Why would you recommend Exiqon’s LNA™ enhanced products to colleagues?

Within our group we have now used more than 1500 microarrays based on the Exiqon LNA™ capture probes. Over the years this microRNA detection platform has definitely proven itself, enabling us to answer important research questions and to formulate novel hypotheses to be tested in subsequent experiments.


What would be your advice to colleagues about getting started with microRNA profiling today?

Plan carefully, optimize and standardize as much as possible. Because microRNA expression changes are often rather subtle (small changes in microRNA expression can have great functional implications) it is of great importance to standardize all steps of the procedure (from isolation, to sample preparation and profiling) to make sure you will not introduce small variations yourself. With the advent of deep sequencing technologies a reliable microarray platform is still extremely suited and informative for focused experiments.


In your opinion what is the most important factor for a successful microRNA profiling experiment?

The most important factor for a successful microRNA profiling experiment is standardization. In addition it is important to have a reliable detection platform, good quality samples and a carefully thought over hypothesis.


Briefly describe the experiments that followed on from the microarray profiling.

By microarray analyses of human patient material we determined soft tissue sarcoma (sub)type specific microRNAs. The expression of selected microRNAs was validated by qPCR. We used microRNA overexpression (by using microRNA mimics) and knockdown (by using Exiqon’s miRCURY LNA™ microRNA inhibitors) in soft tissue sarcoma cell lines for our functional studies (such as cell proliferation assays, cell cycle analyses, apoptosis assays and drug sensitivity assays). By target prediction algorithms and literature research we determined candidate microRNA target genes. Immunoblotting and qPCR analyses were used to determine changes in target gene expression upon modulation of microRNA expression. Luciferase reporter constructs were used to identify whether the 3’UTR of the target gene is directly regulated by the microRNA.


Where we can read more about the project and results?

One of our studies has recently been accepted for publication. It described the down-regulation of miR-17-92 and miR-221/222 cluster members in gastrointestinal stromal tumors (GIST). MiR-17/20a/222 overexpression in GIST cell lines severely inhibited cell proliferation, affected cell cycle progression, induced apoptosis and regulated their predicted target genes KIT and ETV1, two essential oncogenes in GIST. Gits CM, et al. Br. J. Cancer 2013 PMID: 23969726


What are the future perspectives for this research?

MicroRNA profiling and the functional characterization of microRNAs gave more insight into the role of microRNAs in the tumor biology of soft tissue sarcoma. We identified microRNAs that can potentially be used as diagnostic, prognostic, or predictive biomarkers in soft tissue sarcomas, or could serve as a target for therapy, which shows that microRNAs hold great potential for future management of soft tissue sarcomas. Future research should focus on how these microRNAs can be implemented in the clinical management of these tumors.


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