Smad2/3/4 Pathway Contributes to TGF-β-Induced MiRNA-181b Expression to Promote Gastric Cancer Metastasis by Targeting Timp3.

Background/Aims: Transforming growth factor beta (TGF-beta) plays a major role in tumorigenesis. MicroRNA-181b (miRNA-181b) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. This study aimed to confirm the relationship of miRNA-181b and the TGF-beta-Smad2/3/4 pathway with the induction of the epithelial-to-mesenchymal transition (EMT) in gastric cancer. Methods: This study investigated the ability of TGF-beta to induce migration by wound healing and transwell invasion assays in human gastric cancer cell lines. miRNA expression was altered using miRNA-181b mimic and inhibitor in the same system. Expression of miRNA-181b, the hypothetical target gene Timp3 and EMT-related markers were analyzed by real time real-time quantitative RT-PCR. Immunoblotting was used to investigate the levels of phospho-Smad2 and Smad4. Dual-luciferase reporter assays were performed to confirm the direct binding of miRNA-181b to Timp3. Results: miRNA-181b was significantly upregulated in response to TGF-beta treatment in gastric cancer cell lines. Overexpression of miR-181b mimic induced an in vitro EMT-like change to a phenotype similar to that following TGF-beta treatment alone and was reversed by miRNA-181b inhibitor. Inhibition of TGF-beta-Smad2/3 signaling with SD-208 significantly attenuated the upregulation of miRNA-181b. Knockdown of Smad4 in gastric cancer cells strongly attenuated the upregulation of miRNA-181b. Moreover, miR-181b was found to directly target the 3' untranslated region (3'UTR) of Timp3 mRNA affecting TGF-beta-induced EMT. Conclusions: Our results elucidate a novel mechanism through which the TGF-beta pathway regulates the EMT of gastric cancer cells by increasing the levels of miRNA-181b to target Timp3 via the Smad2/3/4-dependent pathway. These findings provide insights into the cellular and environmental factors regulating EMT, which may guide future studies on therapeutic strategies targeting these cells. (C) 2016 The Author(s) Published by S. Karger AG, Basel