Large-Scale Proteomic Identification of Targets of Cellular miR-197 Downregulated by Enterovirus A71

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.