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. 2017 Nov 27;7:16422. doi: 10.1038/s41598-017-16616-7

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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MiR-16-5p enhances EV71-induced apoptosis and inhibits virus replication. (a,b) Analysis EV71-induced apoptosis in SK-N-SH cells using flow cytometry. 15 h after SK-N-SH cells were infected with EV71, the cells were harvested and analysed by flow cytometry with Annexin V/PI apoptosis assay kits (a). Annexin V-FITC positive cells were collected form three different replicates for statistical analysis (b). (c) Measurement of caspase-3/7 activity in SK-N-SH cells after EV71 infection. SK-N-SH cells were cultured in 96-well plates and infected with EV71 for 0 and 15 h. Caspase-Glo® 3/7 reagent (Promega) were added according the standard protocol, and the luminescence of each sample was measured using a plate-reading luminometer as directed by the luminometer manufacturer. (d) Western blot analysis for PARP and Caspase 3 in EV71-infected SK-N-SH cells. Cells were harvested at the indicated times points after EV71 infection. Anti-PARP, anti-Caspase 3 and anti-GAPDH antibodies were used to detect the expression levels of PARP, Caspase 3 and GAPDH, respectively. (e,f) Overexpression of synthetic miR-16-5p mimics inhibits EV71 replication. SK-N-SH cells transfected with miR-16-5p mimics or control oligonucleotides. Cells were harvested at the indicated times points after EV71 infection. The expression levels of the EV71 VP1 gene were measured by qRT-PCR (e). The expression of miR-16-5p was determined both in cells transfected with miR-16-5p mimics and control mimics by using qRT-PCR (f). (g) Western bolt analysis the influence on EV71 replication and virus-induced apoptosis by overexpressing miR-16-5p. Cells transfected with miR-16-5p mimics or control oligonucleotides were infected with EV71 and assayed for protein expression of EV71 VP1, PARP, caspase 3, cleaved caspase 3, CCND1 and CCNE1. GAPDH was used as a loading control. (h) Titration of EV71 in infected cells transfected with miR-16-5p mimics or control oligonucleotides. The transfected SK-N-SH cells were harvested after EV71 infection for 15 h. Virus titters in infected and mock infected cells were determined by TCID50 assays. (i) Overexpression of miR-16-5p inhibits cell proliferation in EV71-infected cells. SK-N-SH cells were transfected with miR-16-5p mimics or control oligonucleotides. 36 h after transfection, the cells were infected with EV71 for 15 h. CCK-8 assay was performed to measure proliferation in EV71 infected cells. Data represent the mean ± standard deviation of the optical density (OD) value detected at 450 nm from three independent experiments. Data in panels (b,c, e,f,h and i) are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD).Asterisks denote significant differences between indicated samples (*p < 0.05, **p < 0.01, Student’s t test). The blots in d and g were cropped and the full-length blots were displayed in Supplementary Information file.