title: A common polymorphism within MSLN affects miR-611 binding site and soluble mesothelin levels in healthy people.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  SMRP performance as diagnostic biomarker improved by considering the genotype rs1057147. This polymorphism most likely affects a binding site for miR-611.
authors.affiliation: *Laboratory of Computational Oncology, Centre for Integrative Biology, CIBIO, University of Trento, Trento, Italy; †Department of Biology, University of Pisa, Pisa, Italy; ‡Division of Thoracic Surgery, Cardiac and Thoracic Department, University of Pisa, Pisa, Italy; §Department of Medicine, Vercelli Teaching Hospital, Vercelli, Italy; and ‖Section of Occupational Medicine, School of Medicine and Surgery, University of Pisa, Pisa, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Garritano
authors.forename: Chiara
authors.initials: C
authors.lastname: De Santi
authors.forename: Roberto
authors.initials: R
authors.lastname: Silvestri
authors.forename: Ombretta
authors.initials: O
authors.lastname: Melaiu
authors.forename: Monica
authors.initials: M
authors.lastname: Cipollini
authors.forename: Elisa
authors.initials: E
authors.lastname: Barone
authors.forename: Marco
authors.initials: M
authors.lastname: Lucchi
authors.forename: Roberto
authors.initials: R
authors.lastname: Barale
authors.forename: Luciano
authors.initials: L
authors.lastname: Mutti
authors.forename: Federica
authors.initials: F
authors.lastname: Gemignani
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bonotti
authors.forename: Rudy
authors.initials: R
authors.lastname: Foddis
authors.forename: Alfonso
authors.initials: A
authors.lastname: Cristaudo
authors.forename: Stefano
authors.initials: S
authors.lastname: Landi
mesh_terms: D001665:Binding Sites; D016022:Case-Control Studies; D058851:GPI-Linked Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism; D000090204:Mesothelin; D008654:Mesothelioma / Q000235:genetics* / Q000378:metabolism; D000086002:Mesothelioma, Malignant; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide
chemical_list: D058851:GPI-Linked Proteins; C000715527:MSLN protein, human; D035683:MicroRNAs; D000090204:Mesothelin