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 fun...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5268774?pdf=render |
| _version_ | 1819159347488358400 |
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| author | Chiara De Santi Sebastian Vencken Jonathon Blake Bettina Haase Vladimir Benes Federica Gemignani Stefano Landi Catherine M Greene |
| author_facet | Chiara De Santi Sebastian Vencken Jonathon Blake Bettina Haase Vladimir Benes Federica Gemignani Stefano Landi Catherine M Greene |
| author_sort | Chiara De Santi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-22T16:39:07Z |
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| id | doaj.art-2ec5d4916e7443b0a59157e504f668b6 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T16:39:07Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-2ec5d4916e7443b0a59157e504f668b62022-12-21T18:19:54ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01121e017099910.1371/journal.pone.0170999Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing.Chiara De SantiSebastian VenckenJonathon BlakeBettina HaaseVladimir BenesFederica GemignaniStefano LandiCatherine M GreeneMicroRNAs (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.http://europepmc.org/articles/PMC5268774?pdf=render |
| spellingShingle | Chiara De Santi Sebastian Vencken Jonathon Blake Bettina Haase Vladimir Benes Federica Gemignani Stefano Landi Catherine M Greene Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing. PLoS ONE |
| title | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing. |
| title_full | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing. |
| title_fullStr | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing. |
| title_full_unstemmed | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing. |
| title_short | Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing. |
| title_sort | identification of mir 21 5p as a functional regulator of mesothelin expression using microrna capture affinity coupled with next generation sequencing |
| url | http://europepmc.org/articles/PMC5268774?pdf=render |
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