Systematic evaluation of the microRNAome through miR-CATCHv2.0 identifies positive and negative regulators of BRAF-X1 mRNA

Here we present miR-CATCHv2.0, an implemented experimental method that allows the identification of the microRNA species directly bound to an RNA of interest. After cross-linking of microRNA::RNA::Ago2 complexes using formaldehyde, the RNA is fragmented using sonication and then subjected to affinity purification using two sets of biotinylated tiling probes (ODD and EVEN). Finally, enriched microRNA species are retrieved by means of small RNA sequencing coupled with an ad hoc analytical workflow. In BRAFV600E mutant A375 melanoma cells, miR-CATCHv2.0 allowed us to identify 20 microRNAs that target X1, the most abundant isoform of BRAF mRNA. These microRNAs fall into different functional classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and to the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). microRNA-induced variations in BRAFV600E protein levels are most of the times coupled to consistent variations in pMEK levels, in melanoma cell proliferation in vitro and in sensitivity to the BRAF inhibitor vemurafenib in a xenograft model in zebrafish. However, microRNAs exist that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein. Our study proposes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and, by using such a tool, unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells.

本报告展示了miR-CATCHv2.0的实现实验方法，该方法能够直接识别与目标RNA直接结合的microRNA物种。通过使用甲醛交联microRNA::RNA::Ago2复合物，RNA经过超声破碎后，利用两组生物素化探针（ODD和EVEN）进行亲和纯化。最终，通过结合小RNA测序及特定的分析工作流程，富集的microRNA物种被成功获取。在BRAFV600E突变型A375黑色素瘤细胞中，miR-CATCHv2.0使我们能够识别出20种靶向BRAF mRNA最丰富同质体X1的microRNA。这些microRNA根据它们所发挥的作用（降低/增加BRAFV600E mRNA和蛋白水平）及其实现这一作用的机制（X1 mRNA的稳定性破坏/稳定或其翻译的降低/增加）而归属于不同的功能类别。microRNA诱导的BRAFV600E蛋白水平变化通常伴随着pMEK水平的持续变化，以及在体外黑色素瘤细胞增殖和斑马鱼异种移植模型中对BRAF抑制剂vemurafenib敏感性的变化。然而，存在某些microRNA能够将ERK途径激活程度与BRAFV600E蛋白水平解耦。本研究提出miR-CATCHv2.0作为识别直接microRNA靶标相互作用的有效工具，并通过使用此工具，揭示了BRAFV600E和ERK途径在黑色素瘤细胞中受后转录调控的复杂性。