Quantification and Localization of Protein-RNA Interactions in Patient-Derived Archival Tumor Tissue
Quantification and Localization of Protein-RNA Interactions in Patient-Derived Archival Tumor Tissue
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DOI：
10.1158/0008-5472.can-19-1094
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发表时间：
2019-10-15
期刊：
影响因子：
11.2
通讯作者：
Santangelo, Philip J.
中科院分区：
文献类型：
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作者：
Blanchard, Emmeline L.;Argyropoulou, Danae;Santangelo, Philip J.
由mRNA-蛋白质相互作用的改变诱导的异常转录后调节在肿瘤发生和癌症进展期间是至关重要的，并且是癌细胞的标志。需要更全面的了解来开发治疗方法和预测结果。细胞和小鼠肿瘤模型不足以进行有力的研究，因为它们缺乏一致性和对人类的可翻译性。此外，迄今为止，在人肿瘤组织中的研究主要限于蛋白质和mRNA的表达分析，其不一定提供关于mRNA-蛋白质相互作用的频率的信息。在这里，我们展示了一种新的优化方法，该方法基于FISH和邻近连接技术，以量化与存档患者来源的肿瘤组织中肿瘤发生和癌症进展相关的RNA结合蛋白的mRNA相互作用。该方法在几种细胞模型和多种类型的存档人类肿瘤样品中针对多个mRNA-蛋白质对进行了验证。此外，这种方法允许通过肿瘤微阵列对广泛的肿瘤类型和阶段的mRNA-蛋白质相互作用进行高通量分析。该方法具有定量、特异性和灵敏度，可用于检测单个细胞和全组织规模的相互作用及其定位，具有单一相互作用灵敏度。这项工作为高通量研究癌症转录后调控提供了一个重要工具，具有很大的潜力，可用于任何感兴趣的mRNA-蛋白质相互作用的应用。这项工作提出了一种方法，敏感，具体，并定量检测和定位天然mRNA和蛋白质相互作用，用于分析患者中的异常转录后调节，来源的存档肿瘤样品。
Abnormal post-transcriptional regulation induced by alterations of mRNA-protein interactions is critical during tumorigenesis and cancer progression and is a hallmark of cancer cells. A more thorough understanding is needed to develop treatments and foresee outcomes. Cellular and mouse tumor models are insufficient for vigorous investigation as they lack consistency and translatability to humans. Moreover, to date, studies in human tumor tissue are predominately limited to expression analysis of proteins and mRNA, which do not necessarily provide information about the frequency of mRNA-protein interactions. Here, we demonstrate novel optimization of a method that is based on FISH and proximity ligation techniques to quantify mRNA interactions with RNA-binding proteins relevant for tumorigenesis and cancer progression in archival patient-derived tumor tissue. This method was validated for multiple mRNA-protein pairs in several cellular models and in multiple types of archival human tumor samples. Furthermore, this approach allowed high-throughput analysis of mRNA-protein interactions across a wide range of tumor types and stages through tumor microarrays. This method is quantitative, specific, and sensitive for detecting interactions and their localization at both the individual cell and whole-tissue scales with single interaction sensitivity. This work presents an important tool in investigating post-transcriptional regulation in cancer on a high-throughput scale, with great potential for translatability into any applications where mRNA-protein interactions are of interest.Significance: This work presents an approach to sensitively, specifically, and quantitatively detect and localize native mRNA and protein interactions for analysis of abnormal post-transcriptional regulation in patient-derived archival tumor samples.