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首页> 外文期刊>BMC Genomics >A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence
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A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence

机译：一种典型的非恶性上皮模型，用于研究基因组动力学并在致癌基因诱导的衰老过程中同时监测微RNA和蛋白质

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Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now. In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs. This experimental setting has three advantages that are presented and discussed: i) it covers a “gap” in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics. Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.
机译：衰老是涉及包括癌症在内的各种病理的基本生物学过程。关于致癌作用，至少在其初始阶段，衰老表示抗肿瘤反应，需要抗癌反应才能使其进展。微小RNA是调节性非编码RNA的子类，参与衰老调节。在亚细胞水平上，与蛋白质相似的微RNA已显示在影响细胞行为的细胞器之间运输。相对于它们的亚细胞定位及其在衰老生物学中的作用，微RNA的差异功能提高了同时进行衰老细胞中编码和非编码基因产物的原位分析的必要性。然而，迄今为止，技术挑战使得原位共检测不可行。在本报告中，我们描述了一种利用新的市售SenTraGorTM化合物绕过这些技术局限性的方法，首次实现了在细胞衰老的生物学背景下同时检测微RNA和蛋白质。该方法被用于我们为研究目的而生成的致癌基因诱导的衰老的典型人类非恶性上皮模型。为了表征这个新颖的系统，我们应用了广泛的细胞和分子技术，以及转录组和微小RNA的高通量分析。该实验装置具有三个优点，这些优点已被介绍和讨论：i）它涵盖了分子致癌领域的“空白”，因为几乎所有相应的体外模型都是基于成纤维细胞的，尽管大多数肿瘤都具有上皮起源，ii）它根据自然选择在很短的时间内概括了上皮肿瘤发生的癌前期和癌期。iii）复制许可因子CDC6用作致癌信号，过表达时与DNA复制和转录有关，可用于监测RNA动态的特征。因此，我们证明了我们的模型对于研究上皮癌变的分子基础是最理想的，该分子基础为肿瘤引发事件提供了依据。后者可能揭示具有临床益处的新型分子靶标。此外，由于该方法可以结合各种基于低，中或高通量图像的方法，因此我们希望它可以广泛应用。

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