一组微RNA基因的甲基化作为非小细胞肺癌诊断和预后的标志物

V. Loginov, M. Gubenko, A. Burdennyy, I. Pronina, P. Postnikov, Yu. A. Efimova, F. V. Radus, E. S. Mochalova, T. Kazubskaya
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One of the approaches for regulating the expression of microRNAs themselves is to change the methylation of the CpG island adjacent to the microRNA gene or overlapping it. It has been shown that microRNA genes are several times more likely to undergo methylation than protein-coding genes. The aim of the present work is to study changes in the level of methylation of a number of microRNA genes and compile a potential panel of markers for the diagnosis and prognosis of NSCLC.Methods. Samples of NSCLC tumors were collected and clinically characterized at the Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation, Moscow, Russia. High-molecular-weight DNA was isolated from tissues using a standard method. The level of methylation was analyzed using bisulfite conversion of DNA and quantitative methyl-specific polymerase chain reaction with real-time detection. The significance of differences between the studied groups was assessed by the nonparametric Mann–Whitney U test for independent samples. Differences were considered significant at p < 0.05.Results. The analysis of methylation levels of microRNA genes revealed a significant (p < 0.05) increase in the methylation level of eight microRNA genes: MIR124-1/2/3, MIR125В-1, MIR129-2, MIR137, MIR375, MIR1258, and MIR339 (p < 0.01, false discovery rate ≤ 0.25). On the basis of receiver operating characteristic analysis, a panel of markers is proposed for the diagnosis of NSCLC according to the nature of methylation of the studied microRNA genes in the tumor and in the normal tissue.Conclusions. 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摘要

目的。肺癌是一种难以诊断的异质性恶性肿瘤,其特点是晚期无症状、不良后果发生率高、转移概率高。其最常见的形式是非小细胞肺癌(NSCLC)。最近的研究表明,非编码 RNA(尤其是 microRNA)在 NSCLC 的发展过程中起着重要作用。微RNA是蛋白质编码基因(包括与肿瘤发生相关的基因)表达的转录后调节因子,参与细胞增殖、分化和凋亡过程。调节 microRNA 自身表达的方法之一是改变与 microRNA 基因相邻或重叠的 CpG 岛的甲基化。研究表明,microRNA 基因发生甲基化的几率是蛋白质编码基因的几倍。本研究的目的是研究一些 microRNA 基因甲基化水平的变化,并为 NSCLC 的诊断和预后编制一个潜在的标记物小组。NSCLC肿瘤样本由俄罗斯联邦卫生部布洛金国家肿瘤医学研究中心(位于俄罗斯莫斯科)收集并进行临床鉴定。采用标准方法从组织中分离出高分子量 DNA。利用 DNA 的亚硫酸氢盐转化和实时检测的定量甲基特异性聚合酶链反应分析甲基化水平。研究组间差异的显著性通过独立样本非参数曼-惠特尼 U 检验进行评估。P<0.05为差异显著。对 microRNA 基因甲基化水平的分析表明,8 个 microRNA 基因的甲基化水平显著增加(p < 0.05):MIR124-1/2/3、MIR125В-1、MIR129-2、MIR137、MIR375、MIR1258和MIR339(P<0.01,误发现率≤0.25)。在接受者操作特征分析的基础上,根据所研究的microRNA基因在肿瘤和正常组织中甲基化的性质,提出了一组用于诊断NSCLC的标记物。我们的研究结果有助于了解 NSCLC 发病的分子机制,可用于开发新的临床肿瘤学诊断和预后方法。
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Methylation of a group of microRNA genes as a marker for the diagnosis and prognosis of non-small cell lung cancer
Objectives. Lung cancer, representing a difficult-to-diagnose heterogeneous malignant neoplasm, is characterized by an asymptomatic course up to late stages, a high incidence of adverse outcomes, and a high probability of metastasis. Its most common form is non-small cell lung cancer (NSCLC). Recent studies have demonstrated a significant role of non-coding RNAs—in particular, microRNAs—in the development of NSCLC. MicroRNAs, which function as post-transcriptional regulators of the expression of protein-coding genes, including those associated with oncogenesis, are involved in the processes of cell proliferation, differentiation, and apoptosis. One of the approaches for regulating the expression of microRNAs themselves is to change the methylation of the CpG island adjacent to the microRNA gene or overlapping it. It has been shown that microRNA genes are several times more likely to undergo methylation than protein-coding genes. The aim of the present work is to study changes in the level of methylation of a number of microRNA genes and compile a potential panel of markers for the diagnosis and prognosis of NSCLC.Methods. Samples of NSCLC tumors were collected and clinically characterized at the Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation, Moscow, Russia. High-molecular-weight DNA was isolated from tissues using a standard method. The level of methylation was analyzed using bisulfite conversion of DNA and quantitative methyl-specific polymerase chain reaction with real-time detection. The significance of differences between the studied groups was assessed by the nonparametric Mann–Whitney U test for independent samples. Differences were considered significant at p < 0.05.Results. The analysis of methylation levels of microRNA genes revealed a significant (p < 0.05) increase in the methylation level of eight microRNA genes: MIR124-1/2/3, MIR125В-1, MIR129-2, MIR137, MIR375, MIR1258, and MIR339 (p < 0.01, false discovery rate ≤ 0.25). On the basis of receiver operating characteristic analysis, a panel of markers is proposed for the diagnosis of NSCLC according to the nature of methylation of the studied microRNA genes in the tumor and in the normal tissue.Conclusions. Our results, which contribute to the understanding of molecular mechanisms involved in NSCLC development, can be used in the development of new diagnostic and prognostic approaches in clinical oncology.
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