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

Date Published: January 26, 2017

Publisher: Public Library of Science

Author(s): Chiara De Santi, Sebastian Vencken, Jonathon Blake, Bettina Haase, Vladimir Benes, Federica Gemignani, Stefano Landi, Catherine M. Greene, Fabio Martelli.

http://doi.org/10.1371/journal.pone.0170999

Abstract

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.

Partial Text

MicroRNAs (miRNAs) are small non-coding RNA molecules, 20–25 nucleotides long, highly conserved in the plant and animal world. They play a major role in post-transcriptional regulation, mainly silencing target mRNAs by binding to miRNA recognition elements (MREs) in the 3’untranslated region (3’UTR) and thus decreasing their corresponding protein levels. Although the basic mechanisms underlying their biogenesis and function are mostly known, the identification of bona fide targets of miRNAs represents the most challenging aspect for researchers in this field. Recently, different methods have been developed to identify multiple miRNAs binding to a single mRNA of interest [1,2]. Since these methods are limited to the 3’UTR of the mRNA, miRNAs targeting the 5’UTR or the coding sequence cannot be identified.

In this work we applied the previously published miR-CATCH protocol [3] to MSLN mRNA in order to identify miRNAs targeting this transcript. With this technique, MSLN mRNA was successfully isolated using MSLN_3 oligo and its cognate miRNAs were identified by next generation sequencing. In vitro experimental analyses validated the findings suggested by NGS, namely that miR-21-5p is a regulator of MSLN mRNA.

In conclusion, this work shows the ability of the miR-CATCH method to generate suitable samples for high-throughput analysis by Next Generation Sequencing. Followed by further experimental validation of miRNA::mRNA interaction, this technique identified miR-21-5p as a regulator of MSLN mRNA that may have role in its increased expression and in the proliferation of tumour cells in MPM.

 

Source:

http://doi.org/10.1371/journal.pone.0170999

 

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