- 作者：Takayuki Muraoka ; Junichi Soh ; 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
- 关键词：Digital PCR ; Malignant pleural mesothelioma ; microRNA ; miR-34b/c ; Methylation ; Circulating DNA
- 刊名：Lung Cancer
- 出版年：December, 2013
- 年：2013
- 卷：82
- 期：3
- 页码：485-490
- 全文大小：845 K
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 掳C which was the mean 卤 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.