title: Smad2/3/4 Pathway Contributes to TGF-β-Induced MiRNA-181b Expression to Promote Gastric Cancer Metastasis by Targeting Timp3.
abstract: OBJECTIVE
  Transforming growth factor beta (TGF-β) 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-β-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-β 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-β 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-β treatment alone and was reversed by miRNA-181b inhibitor. Inhibition of TGF-β-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-β-induced EMT.
  
  
  CONCLUSIONS
  Our results elucidate a novel mechanism through which the TGF-β 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.
authors.affiliation: Department of Tumor Biological Treatment, Changzhou, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhou
authors.forename: Xiao
authors.initials: X
authors.lastname: Zheng
authors.forename: Lujun
authors.initials: L
authors.lastname: Chen
authors.forename: Bin
authors.initials: B
authors.lastname: Xu
authors.forename: Xin
authors.initials: X
authors.lastname: Yang
authors.forename: Jingting
authors.initials: J
authors.lastname: Jiang
authors.forename: Changping
authors.initials: C
authors.lastname: Wu
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008856:Microscopy, Fluorescence; D009362:Neoplasm Metastasis; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics*; D051785:Smad Proteins / Q000235:genetics* / Q000378:metabolism; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D020413:3' Untranslated Regions; C540193:MIrn181 microRNA, human; D035683:MicroRNAs; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D016212:Transforming Growth Factor beta