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. 2017 Jun 27;7:4281. doi: 10.1038/s41598-017-04368-3

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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-224 modulated expression of USP3 and SMAD4. (a) miR-224 mimics attenuated protein expression of USP3 and SMAD4 and (b) miR-224 inhibitor enhanced USP3 and SMAD4 expression. Western blots are cropped; full-length blots are included in Supplementary Figures S15 and S16. The results were quantified by ImageJ software, and the relative protein ratio was normalized to β-actin. (c) Expression of USP3 and SMAD4 mRNA were reduced by miR-224 mimics in LoVo and HCT116 cells. (d,e) Left upper panel: Schematic diagram of miR-224-binding sites in the SMAD4 or the USP3 3′UTRs from microRNA.org. Left lower panel: Construction of USP3 or SMAD4 luciferase reporter plasmids containing wild-type miR-224 MREs (WT) or mutant sequences (MUT). Right panel: Luciferase reporters harbouring wild-type or mutant miR-224-binding sites of the USP3/SMAD4 3′UTR were co-transfected with miR-224 mimics/mi-NC. Relative luciferase activity is presented as the mean ± SD (n = 3). (f,g) miR-224 expression was determined by qRT-PCR after USP3/SMAD4 silencing or USP3/SMAD4 CDS or 3′UTR overexpression in CRC cells. *P < 0.05.