The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma

Takayuki Muraoka, Junichi Sou, Shinichi Toyooka, Keisuke Aoe, Nobukazu Fujimoto, Shinsuke Hashida, Yuho Maki, Norimitsu Tanaka, Kazuhiko Shien, Masashi Furukawa, Hiromasa Yamamoto, Hiroaki Asano, Kazunori Tsukuda, Takumi Kishimoto, Takemi Otsuki, Shinichiro Miyoshi

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Objectives: 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). Materials and 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.

Original languageEnglish
Pages (from-to)485-490
Number of pages6
JournalLung Cancer
Volume82
Issue number3
DOIs
Publication statusPublished - Dec 2013

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MicroRNAs
Methylation
DNA
Serum
Polymerase Chain Reaction
Pleurisy
Asbestos
ROC Curve
Freezing
Healthy Volunteers
Malignant Mesothelioma
Temperature
DNA Methylation
Down-Regulation

Keywords

  • Circulating DNA
  • Digital PCR
  • Malignant pleural mesothelioma
  • Methylation
  • MicroRNA
  • MiR-34b/c

ASJC Scopus subject areas

  • Oncology
  • Pulmonary and Respiratory Medicine
  • Cancer Research

Cite this

The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma. / Muraoka, Takayuki; Sou, Junichi; Toyooka, Shinichi; Aoe, Keisuke; Fujimoto, Nobukazu; Hashida, Shinsuke; Maki, Yuho; Tanaka, Norimitsu; Shien, Kazuhiko; Furukawa, Masashi; Yamamoto, Hiromasa; Asano, Hiroaki; Tsukuda, Kazunori; Kishimoto, Takumi; Otsuki, Takemi; Miyoshi, Shinichiro.

In: Lung Cancer, Vol. 82, No. 3, 12.2013, p. 485-490.

Research output: Contribution to journalArticle

Muraoka, T, Sou, J, Toyooka, S, Aoe, K, Fujimoto, N, Hashida, S, Maki, Y, Tanaka, N, Shien, K, Furukawa, M, Yamamoto, H, Asano, H, Tsukuda, K, Kishimoto, T, Otsuki, T & Miyoshi, S 2013, 'The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma', Lung Cancer, vol. 82, no. 3, pp. 485-490. https://doi.org/10.1016/j.lungcan.2013.09.017
Muraoka, Takayuki ; Sou, Junichi ; Toyooka, Shinichi ; Aoe, Keisuke ; Fujimoto, Nobukazu ; Hashida, Shinsuke ; Maki, Yuho ; Tanaka, Norimitsu ; Shien, Kazuhiko ; Furukawa, Masashi ; Yamamoto, Hiromasa ; Asano, Hiroaki ; Tsukuda, Kazunori ; Kishimoto, Takumi ; Otsuki, Takemi ; Miyoshi, Shinichiro. / The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma. In: Lung Cancer. 2013 ; Vol. 82, No. 3. pp. 485-490.
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T1 - The degree of microRNA-34b/c methylation in serum-circulating DNA is associated with malignant pleural mesothelioma

AU - Muraoka, Takayuki

AU - Sou, Junichi

AU - Toyooka, Shinichi

AU - Aoe, Keisuke

AU - Fujimoto, Nobukazu

AU - Hashida, Shinsuke

AU - Maki, Yuho

AU - Tanaka, Norimitsu

AU - Shien, Kazuhiko

AU - Furukawa, Masashi

AU - Yamamoto, Hiromasa

AU - Asano, Hiroaki

AU - Tsukuda, Kazunori

AU - Kishimoto, Takumi

AU - Otsuki, Takemi

AU - Miyoshi, Shinichiro

PY - 2013/12

Y1 - 2013/12

N2 - Objectives: 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). Materials and 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.

AB - Objectives: 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). Materials and 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.

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KW - Digital PCR

KW - Malignant pleural mesothelioma

KW - Methylation

KW - MicroRNA

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