Purification and Identification of miRNA Target Sites in Genome Using DNA Affinity Precipitation
Purification and Identification of miRNA Target Sites in Genome Using DNA Affinity Precipitation
复制标题
使用 DNA 亲和沉淀纯化和鉴定基因组中的 miRNA 靶位点
DOI：
10.3389/fgene.2019.00778
复制
发表时间：
2019-09-12
影响因子：
3.7
通讯作者：
Xiang，Shuanglin
中科院分区：
文献类型：
--
作者：
Xun，Yu;Tang，Yinxing;Xiang，Shuanglin
与基因组DNA的结合是microRNA（miRNA）执行生物过程的重要方式之一。然而，由于缺乏实验方法，所鉴定的microRNA靶向的基因组位点仅位于启动子和增强子区域。本研究基于对miRNA 3′端标记生物素的亲和纯化，建立了一种高效的体内筛选全基因组区域miRNA结合序列的实验方法。生物素化的 miR-373 用于在 MCF-7 细胞中测试我们的方法，然后使用 Sanger 和下一代测序来筛选 miR-373 结合序列。我们的结果表明，miR-373沉淀的基因组片段不仅位于启动子中，还位于内含子、外显子和基因间。选择 11 个潜在的 miR-373 靶基因进行进一步研究，所有这些基因均受到 miR-373 的显着调控。此外，位于E-钙粘蛋白、冷休克结构域蛋白C2（CSDC2）和PDE4D基因中的靶向序列可以与MCF-7细胞中的miR-373相互作用，而不是HeLa细胞中的miR-373，这与我们的数据一致，即这三个基因可以在MCF-7细胞中受到miR-373的调节，而在HeLa细胞中则不受miR-373的调节。总体而言，这是一种识别全基因组中miRNA靶向序列的有效方法。
Combination with genomic DNA is one of the important ways for microRNAs (miRNAs) to perform biological processes. However, because of lack of an experimental method, the identified genomic sites targeted by microRNA were only located in the promoter and enhancer regions. In this study, based on affinity purification of labeled biotin at the 3′-end of miRNAs, we established an efficiently experimental method to screen miRNA binding sequences in the whole genomic regions in vivo. Biotinylated miR-373 was used to test our approach in MCF-7 cells, and then Sanger and next-generation sequencing were used to screen miR-373 binding sequences. Our results demonstrated that the genomic fragments precipitated by miR-373 were located not only in promoter but also in intron, exon, and intergenic. Eleven potentially miR-373 targeting genes were selected for further study, and all of these genes were significantly regulated by miR-373. Furthermore, the targeting sequences located in E-cadherin, cold-shock domain-containing protein C2 (CSDC2), and PDE4D genes could interact with miR-373 in MCF-7 cells rather than HeLa cells, which is consistent with our data that these three genes can be regulated by miR-373 in MCF-7 cells while not in HeLa cells. On the whole, this is an efficient method to identify miRNA targeting sequences in the whole genome.