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

Our plain-English summary of A common polymorphism within MSLN affects miR-611 binding site and soluble mesothelin levels in healthy people, by S. Garritano, C. De Santi, R. Silvestri, et al (Journal of Thoracic Oncology, 2014).

The study

• Authors: S. Garritano, C. De Santi, R. Silvestri, O. Melaiu, M. Cipollini, E. Barone, M. Lucchi, R. Barale, L. Mutti, F. Gemignani, A. Bonotti, R. Foddis, A. Cristaudo, S. Landi
• Journal: Journal of Thoracic Oncology
• Published: 2014;9(11):1662-8
• Cited by: 20 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

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.

In plain English

This study asked whether a common DNA variant, rs1057147, in the mesothelin gene affects blood levels of soluble mesothelin-related peptide (SMRP), a proposed marker for pleural mesothelioma. Participants were 759 asbestos-exposed workers in a surveillance program in Pisa, 2009 to 2012: 371 healthy, 318 with benign respiratory diseases and 70 with pleural mesothelioma. The variant was not associated with mesothelioma risk (odds ratio 0.91; 95% confidence interval 0.53 to 1.55). Among people without mesothelioma, average SMRP rose with the number of A copies (GG 0.89, GA 1.14, AA 1.71 nM); mesothelioma patients showed no such pattern. Comparing all mesothelioma patients with everyone else, an SMRP cutoff of 1.28 nM gave 79.7% specificity (63.9% at 1 nM). Using only GG people without mesothelioma as the comparison group, specificity at 1.28 nM was 85.3%. In cell experiments, the regulatory molecule miR-611 acted only on the G version. The authors concluded that considering genotype improved SMRP's performance and that the variant most likely affects a miR-611 binding site. They noted few AA carriers and no data on glomerular filtration rate, a kidney measure.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Genetic association study in an asbestos-exposed surveillance population, combined with laboratory experiments on cell lines and pleural tissue samples
• Who was studied: 759 people (371 healthy, 318 with benign respiratory diseases, 70 with malignant pleural mesothelioma) recruited 2009 to 2012 at the University Hospital of Pisa, Italy, through an occupational surveillance program for workers previously exposed to asbestos. Separately, the laboratory work used 14 mesothelioma and 14 healthy pleural biopsies and two cell lines (Met-5A, Mero-14).
• Exposure: All participants had past asbestos exposure (fiber types not stated). The factor studied was each person's genotype at rs1057147 (G or A) in the MSLN gene, in relation to serum SMRP levels.
• Main results:
  • No association between the rs1057147 variant and risk of mesothelioma when comparing GA plus AA with GG genotypes: odds ratio 0.91, 95% confidence interval 0.53 to 1.55, p = 0.72
  • Mean serum SMRP was 3.58 nM in mesothelioma patients, compared with 0.94 in healthy subjects and 1.04 in those with benign respiratory diseases (p less than 0.0001)
  • Among mesothelioma patients, SMRP levels did not differ significantly by genotype (p = 0.658)
  • Among non-mesothelioma subjects (healthy plus benign disease), mean SMRP increased with the number of A alleles: GG 0.89 (n = 468), GA 1.14 (n = 198), AA 1.71 (n = 23); p less than 0.0001
  • Comparing all mesothelioma patients with all non-mesothelioma subjects, without genotype, an SMRP cutoff of 1.28 nM gave a sensitivity of 75.7% and a specificity of 79.7%
  • In the same comparison at a cutoff of 1 nM, sensitivity was 87.1% and specificity was 63.9%
  • For the genotype analysis, all mesothelioma patients formed the diseased group and the non-mesothelioma comparison group was split by genotype
  • When only non-mesothelioma subjects with GG genotype were the comparison group, at a cutoff of 1.28 nM the AUC was 0.888 and specificity was 85.3%
  • At 1.28 nM, specificity was lower when the comparison group was non-mesothelioma subjects with GA (70.7%) or AA (43.5%) genotypes
  • In Met-5A cells without added miRNA, reporter activity with the G allele was reduced to about 70% of that with the A allele (p less than 0.0001)
  • With the G-allele construct, reporter activity fell to 80% with miR-611 and to 70% with miR-887
  • With the A-allele construct, only miR-887 reduced reporter activity (to 70%). miR-611 caused no change.
  • In Mero-14 mesothelioma cells at 30 nM, mesothelin protein fell to 64% with miR-887 and to 66% with miR-611
  • At 30 nM the reduction was statistically significant for miR-611 (p = 0.016) but not for miR-887 (p = 0.077)
  • Expression of the two miRNAs did not differ significantly between normal and mesothelioma pleural tissue
• The authors' conclusion: The authors concluded that SMRP's performance as a diagnostic marker improved when rs1057147 genotype was considered, and that the variant most likely affects a binding site for miR-611.
• Limitations the authors state:
  • The A allele is relatively rare, so there were too few AA homozygotes for robust statistical analysis, especially in subgroups
  • Other information potentially linked to SMRP levels, such as glomerular filtration rate, could not be ascertained

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelin, Soluble Mesothelin-Related Peptides (SMRP), Pleural Mesothelioma, MeT-5A Cells, Biomarker.

Related studies

• Polymorphisms in the putative micro-RNA-binding sites of mesothelin gene are associated with serum levels of mesothelin-related protein — A. Cristaudo, R. Foddis, et al. (2010)
• Mesothelin promoter variants are associated with increased soluble mesothelin-related peptide levels in asbestos-exposed individuals — C. De Santi, P. Pucci, et al. (2017)
• Two novel polymorphisms in 5' flanking region of the mesothelin gene are associated with soluble mesothelin-related peptide (SMRP) levels — A. Cristaudo, R. Foddis, et al. (2011)
• Mesothelin (MSLN) methylation and soluble mesothelin-related protein levels in a Chinese asbestos-exposed population — M. Yu, Y. Zhang, et al. (2015)
• Evaluation of soluble mesothelin-related peptides and MSLN genetic variability in asbestos-related diseases — K. Goricar, V. Kovac, et al. (2020)
• Association of MiR-126 with soluble mesothelin-related peptides, a marker for malignant mesothelioma — L. Santarelli, E. Strafella, et al. (2011)

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