title: The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma.
abstract: OBJECTIVE
  Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. microRNA-34b/c (miR-34b/c), which plays an important role in the pathogenesis of MPM, is frequently downregulated by DNA methylation in approximately 90% of MPM cases. In this study, we estimated the degree of miR-34b/c methylation in serum-circulating DNA using a digital methylation specific PCR assay (MSP).
  
  
  METHODS
  A real-time MSP assay was performed using the SYBR Green method. The melting temperature (Tm) of each PCR product was examined using a melting curve analysis. For a digital MSP assay, 40 wells were analyzed per sample. A total of 110 serum samples from 48 MPM cases, 21 benign asbestos pleurisy (BAP) cases, and 41 healthy volunteers (HVs) were examined.
  
  
  RESULTS
  Positive range of Tm value for miR-34b/c methylation was defined as 77.71-78.79 °C which was the mean ± 3 standard deviations of 40 wells of a positive control. The number of miR-34b/c methylated wells was counted per sample according to this criterion. The number of miR-34b/c methylated wells in MPM cases was significantly higher than that in BAP cases (P=0.03) or HVs (P<0.001). Advanced MPM cases tended to have higher number of miR-34b/c methylated wells than early MPM cases. Receiver-operating characteristic (ROC) curve analysis revealed that three number of miR-34b/c methylated wells per sample was the best cut-off of positivity of MPM with a 67% of sensitivity and a 77% specificity for prediction. The area under the ROC curve was 0.77.
  
  
  CONCLUSIONS
  Our digital MSP assay can quantify miR-34b/c methylation in serum-circulating DNA. The degree of miR-34b/c methylation in serum-circulating DNA is associated with MPM, suggesting that this approach might be useful for the establishment of a new detection system for MPM.
authors.affiliation: Department of Thoracic Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Muraoka
authors.forename: Junichi
authors.initials: J
authors.lastname: Soh
authors.forename: Shinichi
authors.initials: S
authors.lastname: Toyooka
authors.forename: Keisuke
authors.initials: K
authors.lastname: Aoe
authors.forename: Nobukazu
authors.initials: N
authors.lastname: Fujimoto
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Hashida
authors.forename: Yuho
authors.initials: Y
authors.lastname: Maki
authors.forename: Norimitsu
authors.initials: N
authors.lastname: Tanaka
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Shien
authors.forename: Masashi
authors.initials: M
authors.lastname: Furukawa
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Asano
authors.forename: Kazunori
authors.initials: K
authors.lastname: Tsukuda
authors.forename: Takumi
authors.initials: T
authors.lastname: Kishimoto
authors.forename: Takemi
authors.initials: T
authors.lastname: Otsuki
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Miyoshi
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000032:analysis* / Q000097:blood; D019175:DNA Methylation; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008654:Mesothelioma / Q000175:diagnosis* / Q000235:genetics; D000086002:Mesothelioma, Malignant; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D009367:Neoplasm Staging; D010997:Pleural Neoplasms / Q000175:diagnosis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D014408:Biomarkers, Tumor; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D004247:DNA
keywords: Circulating DNA; Digital PCR; Malignant pleural mesothelioma; Methylation; miR-34b/c; microRNA