Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3'untranslated region (3'UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs::mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA::MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-21-5p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA::mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth.
Competing Interests: This work was supported by two grants: Science Foundation Ireland 12/TIDA/B2265 and Ministero della Salute-Bando Ricerca Finalizzata 2009 (RF-2009-1529895). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was also possible due to the PI being awarded the European Respiratory Society Rare Disease Award 2013 which was sponsored by GlaxoSmithKline. This commercial entity had no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and/or decision to submit for publication. This does not alter our adherence to PLOS ONE policies on sharing data and materials.