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. 2017 May 3;7:1477. doi: 10.1038/s41598-017-01550-5

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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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Effect of miRNA-139-5p overexpression on RhoA/ROCK machinery in IAS SMCs. (A) Western blot data showing that miRNA-139-5p overexpression in the IAS SMCs decreases the expression of RhoA/ROCK signal transduction machinery proteins which is selectively blocked by the anti-miRNA-139-5p. (A) (upper portion). Western blots comparing the expression levels of RhoA, ROCK1, and ROCK2 before and after miRNA-139-5p, anti-miRNA-139-5p alone, and following miRNA-139-5p + anti-miRNA-139-5p. The lower portion of the panel provides the corresponding quantitative data showing that miRNA-139-5p causes significant (*p < 0.05) downregulation of RhoA and ROCK2 without any significant (p > 0.05) effect on the levels of ROCK1. Conversely, anti-miRNA-139-5p by itself causes significant upregulation of RhoA and ROCK2 (*p < 0.05). (As indicated, RhoA, ROCK1, and ROCK2 expressions were compared with GAPDH.) (B) (upper portion).Western blots comparing the expression levels of p-MYPT1and p-MLC₂₀, before and after miRNA-139-5p, anti-miRNA-139-5p alone, and following miRNA-139-5p + anti-miRNA-139-5p. The lower portion of this panel provides the quantitative data showing that miRNA-139-5p significantly (*p < 0.05) downregulates, while anti-miRNA-139-5p causes significant (*p < 0.05) upregulation of p-MYPT1 and p-MLC₂₀. (As shown, p-MYPT1, and p-MLC₂₀ expressions were compared with MYPT1 and MLC₂₀, respectively.) (C) Model representing the effect of miRNA-139-5p on RhoA/ROCK2 signaling cascade. The IAS smooth muscle is characterized by upregulated RhoA/ROCK signaling which may be either constitutively active or GPCR-activated. Data suggest that higher levels of miRNA-139-5p in the IAS attenuate ROCK2 expression, which in turn leads to activation of MLCP (via decrease in p-MYPT1), and decrease in p-MLC₂₀ and basal tone.