A common polymorphism within MSLN Affects miR-611 binding site and soluble mesothelin levels in healthy people

Garritano, S; De Santi, C; Silvestri, R; Melaiu, O; Cipollini, M; Barone, E; Lucchi, M; Barale, R; Mutti, L; Gemignani, F; Bonotti, A; Foddis, R; Cristaudo, A; Landi, S

Abstract

INTRODUCTION:
Soluble mesothelin related peptide (SMRP) was proposed as a promising diagnostic marker for malignant pleural mesothelioma (MPM). In a previous study, we found that rs1057147 within the 3' untranslated region of MSLN gene was associated with SMRP levels. Thus, we aimed to (1) confirm the previous association on a large series of volunteers and (2) test the hypothesis that the SNP could affect microRNA binding sites.

METHODS:
The association analysis was verified in 759 subjects. Then, in silico predictions highlighted miR-611 and miR-887 as candidate miRNAs binding to the polymorphic site. Thus, chimeric constructs bearing the alternative alleles (G > A) were assayed alone or in cotransfection with the miRNA mimics, with dual luciferase reporter assay in non-MPM Met-5A cells. The miRNAs were also assayed by western blot analysis for their ability to down-regulate endogenous mesothelin in the MPM Mero-14 cell line.

RESULTS:
We confirmed that, among non-MPM volunteers, GG homozygotes have the lowest SMRP levels. When the genotype is taken into account, the specificity of SMRP as biomarker improves from 79.7% to 85.3%. Dual-luciferase assays showed a significantly lower reporter activity when the vector harbored the G allele as compared to A allele. miR-887 mimic caused a reduced reporter activity of vectors harboring A or G alleles, while miR-611 was effective only on the vector harboring the G allele. Transfection of these miRNAs into Mero-14 cells significantly reduced endogenous MSLN protein.

CONCLUSION:
SMRP performance as diagnostic biomarker improved by considering the genotype rs1057147. This polymorphism most likely affects a binding site for miR-611.

Citation

Garritano, S., De Santi, C., Silvestri, R., Melaiu, O., Cipollini, M., Barone, E., …Landi, S. (2014). A common polymorphism within MSLN Affects miR-611 binding site and soluble mesothelin levels in healthy people. Journal of Thoracic Oncology, 9(11), 1662-1668. https://doi.org/10.1097/JTO.0000000000000322

• Journal Article Type: Article
• Online Publication Date: Nov 20, 2015
• Publication Date: Nov 1, 2014
• Deposit Date: Feb 2, 2015
• Journal: Journal of Thoracic Oncology
• Print ISSN: 1556-0864
• Electronic ISSN: 1556-1380
• Publisher: Lippincott, Williams & Wilkins
• Peer Reviewed: Peer Reviewed
• Volume: 9
• Issue: 11
• Pages: 1662-1668
• DOI: https://doi.org/10.1097/JTO.0000000000000322
• Publisher URL: http://dx.doi.org/10.1097/JTO.0000000000000322
• Related Public URLs: http://www.jto.org/
• Additional Information: Funders : Buzzi Foundation (Italy)