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. 2021 Aug 11;6:300. doi: 10.1038/s41392-021-00716-y

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© The Author(s) 2021

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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Fig. 2 — miR-7, 24, 145 and 223 in exosomes directly targeted the S protein and inhibited SARS-CoV-2 replication. a Diagrams of the transfection of synthetic miRNA mimics and measurement of the inhibition of S protein expression and antiviral activity in SARS-CoV-2 replication. b Luciferase reporter assays in HEK293T cells transfected with reporter vectors carrying the WT binding site sequence of miR-7-5p, miR-24-3p, miR-145-5p or miR-223-3p vs the mutated binding site sequence in the presence or absence of the indicated miRNA mimics. c Quantitative analysis of Ago2 protein levels by western blot in HEK293T cells after pulling down with biotinylated control probe (NC), miR-7-5p, miR-24-3p, miR-145-5p or miR-223-3p probe. d Quantitative RT-PCR analysis of S mRNA levels in HEK293T after pulling down with biotinylated control probe (NC), miR-7-5p, miR-24-3p, miR-145-5p or miR-223-3p probe. e Quantitative analysis of Ago2 or IgG protein levels in anti-Ago2 or anti-IgG antibody-immunoprecipitated complex by western blot. f Detection of miR-7-5p, miR-24-3p, miR-145-5p or miR-223-3p and S mRNA in anti-Ago2 or anti-IgG antibody-immunoprecipitated complex by RT-qPCR. g, h, i Cytometric analysis (g), fluorescence images (h) and western blot (i) of GFP-S protein-overexpressing HEK293T cells after treatment with synthetic miR-7-5p, miR-24-3p, miR-145-5p or miR-223-3p individually or in combination (miR-mix). j Efficacy of synthetic miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p in inhibiting the replication of SARS-CoV-2. k Diagrams of the collection of exosomes from cell medium after transfection of synthetic miRNA mimics and measurement of the inhibition of S protein expression and antiviral activity in SARS-CoV-2 replication. l, m, n Cytometric analysis (l), fluorescence images (m) and western blot (n) of GFP-S protein-overexpressing HEK293T cells after treatment with exosomes from cells transfected with synthetic miR-7-5p, miR-24-3p, miR-145-5p or miR-223-3p individually or in combination. o Efficacy of exosomal miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p in inhibiting the replication of SARS-CoV-2. Data are shown as means ± SEMs. Statistical significance was assessed using one-way ANOVA coupled with Bonferroni’s post hoc test (b, g, i, j, l, n, o), or two-tailed unpaired Student’s t-test (d, f). *P < 0.05; **P < 0.01; ***P < 0.001