Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations.
Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations.
复制标题
DOI：
10.3389/fgene.2018.00045
复制
发表时间：
2018
影响因子：
3.7
通讯作者：
Svoboda P
中科院分区：
文献类型：
--
作者：
Brustikova K;Sedlak D;Kubikova J;Skuta C;Solcova K;Malik R;Bartunek P;Svoboda P
microRNA（miRNAs）是一类抑制基因表达的小分子RNA。它们导致许多生理过程和病理。因此，操作miRNA途径的策略是令人感兴趣的，因为它们可以为实验或治疗干预提供工具。这些工具之一可以是通过报告分析的高通量筛选（HTS）鉴定的小化合物。虽然已经在这种高通量筛选中鉴定了许多化合物，但它们的应用潜力仍然难以捉摸。在这里，我们报告了我们的经验，基于细胞的HTS的12,816个化合物的库，以确定miRNA通路调节剂。我们使用了人HeLa和小鼠NIH 3 T3细胞系，其稳定整合或瞬时表达的荧光素酶报告基因被内源性miR-30和let-7 miRNA抑制，并鉴定了163种推定的miRNA抑制剂。我们报告说，通过应激诱导缓解miRNA介导的抑制的化合物是罕见的，我们发现只有两种化合物，可重复地诱导应激颗粒和缓解miRNA靶向的报告抑制。然而，我们已经发现，这种测定类型容易产生荧光素酶报告活性的非特异性（miRNA-非依赖性）刺激物。此外，我们的数据提供了部分支持先前发表的miRNA通路调节剂，最显着的交叉点之间发现蒽环类，多巴胺衍生物，黄酮类，和芪。总之，我们的研究结果强调了适当的阴性对照在开发miRNA途径的小化合物抑制剂中的重要性。这特别涉及验证策略，其将极大地受益于与常规使用的miRNA靶向报告基因测定法根本不同的测定法。
MicroRNAs (miRNAs) are small RNAs repressing gene expression. They contribute to many physiological processes and pathologies. Consequently, strategies for manipulation of the miRNA pathway are of interest as they could provide tools for experimental or therapeutic interventions. One of such tools could be small chemical compounds identified through high-throughput screening (HTS) with reporter assays. While a number of chemical compounds have been identified in such high-throughput screens, their application potential remains elusive. Here, we report our experience with cell-based HTS of a library of 12,816 chemical compounds to identify miRNA pathway modulators. We used human HeLa and mouse NIH 3T3 cell lines with stably integrated or transiently expressed luciferase reporters repressed by endogenous miR-30 and let-7 miRNAs and identified 163 putative miRNA inhibitors. We report that compounds relieving miRNA-mediated repression via stress induction are infrequent; we have found only two compounds that reproducibly induced stress granules and relieved miRNA-targeted reporter repression. However, we have found that this assay type readily yields non-specific (miRNA-independent) stimulators of luciferase reporter activity. Furthermore, our data provide partial support for previously published miRNA pathway modulators; the most notable intersections were found among anthracyclines, dopamine derivatives, flavones, and stilbenes. Altogether, our results underscore the importance of appropriate negative controls in development of small compound inhibitors of the miRNA pathway. This particularly concerns validation strategies, which would greatly profit from assays that fundamentally differ from the routinely employed miRNA-targeted reporter assays.