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. 2018 Aug 22;8:12635. doi: 10.1038/s41598-018-30895-8

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© The Author(s) 2018

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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Role of miR-509 in cell proliferation, c-Abl expression, MEK1/2 and ERK1/2 phosphorylation. (A) HASM cells were transfected with miR-Ctrl or miR-509. One day after transfection, they were stimulated with 10 ng/ml PDGF for additional 1–3 days followed by assessment of cell numbers. Exposure to miR-509 inhibits the PDGF-induced proliferation. Data are mean ± SD (n = 5, **p < 0.01). (B) Treatment with miR-509 does not affect c-Abl protein expression. Blots of HASM cells transfected with either miR-Ctrl or miR-509 for 3 days were probed with antibodies against c-Abl and GAPDH. Data are mean ± SD (n = 5). NS, not significant. (C) HASM cells treated with miR-Ctrl or miR-509 for 3 days, or stable Plk1 (miR-509 resistant) expressing cells were treated with miR-509 for 3 days (rescue cells). Cell extracts were evaluated by immunoblot analysis. Data are mean ± SD (n = 5, **p < 0.01). (D) Representative immunoblots illustrating the role of Plk1 in phosphorylation of MEK1/2 (MEK1: Ser-218/Ser-222; MEK2: Ser-222/Ser-226) and ERK1/2 (ERK1: Thr-202/Tyr-204; ERK2: Thr-185/Tyr-187). HASM cells treated with miR-Ctrl or miR-509, or rescue cells (see above) were stimulated with 10 ng/ml PDGF for 10 min or left unstimulated. MEK1/2 and ERK1/2 phosphorylation in these cells was evaluated by immunoblot analysis. Rescue of Plk1 in miR-509 treated cells restores the PDGF-induced phosphorylation of MEK1/2 and ERK1/2. (E) The phosphorylation levels of MEK1/2 and ERK1/2 in cells stimulated with PDGF are normalized to corresponding unstimulated levels. Data are mean ± SD (n = 7, *p < 0.05; **p < 0.01). (F) Smooth muscle cells were treated with miR-Ctrl or miR-509, or stable Plk1 expressing cells were treated with miR-509. One day after treatment, they were stimulated with 10 ng/ml PDGF for 3 days. The numbers of viable cells were then determined. Rescue of Plk1 in miR-509 treated cells recovers the PDGF-induced proliferation in HASM cells. Data are mean ± SD (n = 5, **p < 0.01). Student’s t-test was used for statistical analysis of (A,B). One-way ANOVA was used for statistical analysis of (C). Two-way ANOVA was used for statistical analysis of (E,F).