Department of Biochemistry and Molecular Biology, College of Medicine , Chang Gung University , Taoyuan 333 , Taiwan.
Research Center for Emerging Viral Infections , Chang Gung University , Taoyuan 333 , Taiwan.
J Proteome Res. 2019 Jan 4;18(1):449-460. doi: 10.1021/acs.jproteome.8b00762. Epub 2018 Oct 29.
MicroRNAs are noncoding RNA species comprising 18-23 nucleotides that regulate host-virus interaction networks. Here, we show that enterovirus A71 infection in human rhabdomyosarcoma (RD) is regulated by miR-197 expression. Transfection of miR-197 mimic into RD cells inhibited virus replication by interfering with the viral RNA synthesis. We employed a combination of mass-spectrometry-based quantitative proteomics with the stable isotope labeling with amino acids in cell culture (SILAC) approach for the identification of the miR-197 target genes in RD cells and to investigate the differential expression of the prospective target proteins. A total of 1822 proteins were repeatedly identified in miR-197-transfected RD cells, 106 of which were predicted to have seed sites by TargetScan. Notably, seven of eight selected genes potentially related to viral replication and immune response were validated as direct miR-197 targets, using a luciferase 3'-untranslated region (UTR) reporter assay. The expression levels of three selected endogenous molecules (ITGAV, ETF1, and MAP2K1/MEK1) were significantly reduced when RD cells were transfected with a miR-197 mimic. Our results provide a comprehensive database of miR-197 targets, which might provide better insights into the understanding of host-virus interaction.
微小 RNA 是由 18-23 个核苷酸组成的非编码 RNA 种类，可调节宿主-病毒相互作用网络。在这里，我们表明肠道病毒 A71 感染人横纹肌肉瘤（RD）受到 miR-197 表达的调节。miR-197 模拟物转染 RD 细胞通过干扰病毒 RNA 合成来抑制病毒复制。我们采用基于质谱的定量蛋白质组学与稳定同位素标记的氨基酸在细胞培养中的方法（SILAC）相结合，鉴定 RD 细胞中 miR-197 的靶基因，并研究潜在靶蛋白的差异表达。在 miR-197 转染的 RD 细胞中重复鉴定了 1822 种蛋白质，其中 106 种被 TargetScan 预测具有种子位点。值得注意的是，使用荧光素酶 3'非翻译区（UTR）报告基因测定法，对 8 个潜在与病毒复制和免疫反应相关的选定基因中的 7 个进行了验证，这些基因被证实为直接的 miR-197 靶标。当 RD 细胞转染 miR-197 模拟物时，三种内源性分子（ITGAV、ETF1 和 MAP2K1/MEK1）的表达水平显著降低。我们的研究结果提供了 miR-197 靶标的综合数据库，这可能为深入了解宿主-病毒相互作用提供更好的见解。
