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

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.

MiR-21 over-expression and Programmed Cell Death 4 down-regulation features malignant pleural mesothelioma

“…miRNA dysregulation has also been found in MPM [ 9 ], possibly involved in carcinogenesis [ 10 ]. In particular, several papers reported miR-21 over-expression in MPM [ 11 – 13 ] and we recently demonstrated its reliability as MPM marker [ 14 ]. However, miRNA quantification is not easily available in all laboratories and it would be preferable to use the more common immuno-cyto-chemistry.…”

miR‐CATCH Identifies Biologically Active miRNA Regulators of the Pro‐Survival Gene XIAP, in Chinese Hamster Ovary Cells

“…We demonstrated the effectiveness of a miR‐CATCH method, used to identify biologically active miRNA regulators of an experimentally proven engineering gene, XIAP, which could subsequently be used for genetic engineering without burdening the cells translational machinery. This method has previously been exploited to identify valid miRNA regulators of various genes such as miR‐21‐5p for mesothelin (MSLN) and miR‐659‐3p for progranulin (GRN) but has never been performed in a CHO cell context.…”

Manipulating microRNAs for the Treatment of Malignant Pleural Mesothelioma: Past, Present and Future

“…Using a mimic to restore expression levels revealed miR-29c-5p to have modest tumor (8,9) miR-16-5p ↓ ↓ None √ √ * Tumor suppressor functions; downregulates CCND1, BCL2, and PD-L1 TS (8,9) miR-17-5p ↓ ↓ High Exp = SS √ -Inhibits migration; targets KCNMA1 TS (10) miR-18a-5p ↑ N.D. High Exp = SS √ -Antimir causes modest growth inhibition; targets PIAS3 OncomiR (11) miR-21-5p N.D. ↑ High Exp = SS √ -Mimic causes modest growth inhibition; targets mesothelin TS? (12)(13)(14) miR-24-3p ↑ ↑ N.D. √ ( √ ) Promotes migration and tumor growth in mice; targets CGN Oncomir (15) miR-29c-5p ↓ N.D. High Exp = LS √ -Mimic inhibits growth and migration; targets DNMT1/3A TS (16) miR-31-5p ↓ ↓ High Exp = SS √ -Mimic inhibits growth and migration; targets PPP6C; role in drug resistance TS? (17)(18)(19) miR-34a-5p ↓ ↓ N.D. √ √ i Lost in genetically modified mouse model; targets c-Met TS (20)(21)(22)(23) miR-34b-3p ↓ ↓ N.D. √ √ Inhibits MPM growth, enhance radiosensitivity; inhibitors promote mesothelial proliferation TS (21,22,(24)(25)(26) suppressor activity in two MPM cell lines in vitro, by inhibition of proliferation and migration/invasion.…”

“…In light of these observations, it is surprising that the only study to date to assess miR-21-5p activity in MPM suggests that it has modest tumor suppressor function. In a study designed to identify regulators of the MPM marker mesothelin (MSLN), both miR-21-5p and miR-100-5p were found to interact with the MSLN 3'UTR (14). Further experimentation revealed that a miR-21-5p mimic led to modest but significant inhibition of proliferation in two MPM cell lines, with a more pronounced reduction in colony forming ability.…”