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.

Garritano, S., De Santi, C., Silvestri, R., Melaiu, O., Cipollini, M., Barone, E., et al. (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 [10.1097/JTO.0000000000000322].

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

Melaiu O.;
2014-01-01

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.
2014
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore MED/05 - PATOLOGIA CLINICA
English
Malignant pleural mesothelioma
Single nucleotide polymorphism
miRSNP
MicroRNA
Soluble mesothelin
related peptide
Garritano, S., De Santi, C., Silvestri, R., Melaiu, O., Cipollini, M., Barone, E., et al. (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 [10.1097/JTO.0000000000000322].
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; Cris...espandi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/305986
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