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. 2019 Nov 26;39(9):1983–1996. doi: 10.1038/s41388-019-1120-z

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© The Author(s) 2019

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. 3 — miR-200a overexpression resulted in C-Jun phosphorylation, and its regulated MMP-2 transcription and protein expression. a, b Total RNA was extracted from cells and MMP-2 mRNA levels evaluated using real-time PCR. Wild-type MMP-2 promoter-driven luciferase reporter was co-transfected together with pRL-TK into T24T(Vector) and T24T(miR-200a) cells (c), or UMUC3(Vector) and UMUC3(miR-200a) cells (d). After 24 h of transfection, luciferase activity was evaluated. TK was used as an internal control. Results presented as MMP-2 promoter activity relative to control vector transfectant. Each bar represents means ± SD of three independent experiments. e Potential transcription factor binding sites in human MMP-2 promoter region. Extracts obtained from T24T(Vector) vs. T24T(miR-200a) (f) or UMUC3(Vector) vs. UMUC3(miR-200a) (g) cells were analyzed for activation/expression of transcription factors indicated. h TAM67 and vector control were stably transfected into T24T(miR-200a) cells and transfectants were identified by western blot. i TAM67 and its vector control were stably transfected into T24T(miR-200a) cells; total RNA was extracted, and MMP-2 mRNA levels were evaluated using real-time PCR). Bars represent means ± SD from three independent experiments. j Wild-type MMP-2 promoter-driven luciferase reporter was co-transfected together with pRL-TK into T24T(miR-200a/Vector) and T24T(miR-200a/TAM67) cells. After 24 h of transfection, cells were extracted for determination of luciferase activity. Results were presented as MMP-2 promoter activity relative to vector control transfectants. Each bar represents means ± SD from three independent experiments. *p < 0.01