miR-300 inhibits epithelial to mesenchymal transition and metastasis by targeting Twist in human epithelial cancer

Mol Cancer. 2014 May 24;13:121. doi: 10.1186/1476-4598-13-121.

Abstract

Background: Epithelial-to-mesenchymal transition (EMT) is a key step of the progression of tumor cell metastasis. Recent work has demonstrated some miRNAs play critical roles in EMT. In this study, we focused on the roles of miR-300 in regulating EMT.

Methods: The expression levels of miR-300 were examined in epithelial carcinoma cells that underwent an EMT using quantitative reverse transcription-PCR. The role of miR-300 in EMT was investigated by transfection of the miR-300 mimic or inhibitor in natural epithelial-mesenchymal phenotype cell line pairs and in transforming growth factor (TGF) beta-induced EMT cell models. A luciferase reporter assay and a rescue experiment were conducted to confirm the target gene of miR-300. The efficacy of miR-300 against tumor invasion and metastasis was evaluated both in vitro and in vivo. Correlation analysis between miR-300 expression and the expression levels of its target gene, as well as tumor metastasis was performed in specimens from patients with head and neck squamous cell carcinoma (HNSCC).

Results: MiR-300 was found down-regulated in the HNSCC cells and breast cancer cells that underwent EMT. Ectopic expression of miR-300 effectively blocked TGF-beta-induced EMT and reversed the phenotype of EMT in HN-12 and MDA-MB-231 cells, but inhibition of miR-300 in the epithelial phenotype cells, HN-4 and MCF-7 cells, could induce EMT. The luciferase reporter assay and the rescue assay results showed that miR-300 directly targets the 3'UTR of Twist. Enforced miR-300 expression suppressed cell invasion in vitro and experimental metastasis in vivo. Clinically, miR-300 expression was found inversely correlated with Twist expression and reduced miR-300 was associated with metastasis in patient specimens.

Conclusions: Down-regulation of miR-300 is required for EMT initiation and maintenance. MiR-300 may negatively regulate EMT by direct targeting Twist and therefore inhibit cancer cell invasion and metastasis, which implicates miR-300 as an attractive candidate for cancer therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Breast Neoplasms / genetics*
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Carcinoma, Squamous Cell / genetics*
  • Carcinoma, Squamous Cell / metabolism
  • Carcinoma, Squamous Cell / pathology
  • Cell Line, Tumor
  • Epigenesis, Genetic
  • Epithelial Cells / metabolism
  • Epithelial Cells / pathology
  • Epithelial-Mesenchymal Transition / genetics*
  • Female
  • Gene Expression Regulation, Neoplastic
  • Genes, Reporter
  • Head and Neck Neoplasms / genetics*
  • Head and Neck Neoplasms / metabolism
  • Head and Neck Neoplasms / pathology
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / metabolism
  • Lung Neoplasms / secondary
  • Mice
  • Mice, Nude
  • MicroRNAs / antagonists & inhibitors
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Neoplasm Invasiveness
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Signal Transduction
  • Twist-Related Protein 1 / genetics*
  • Twist-Related Protein 1 / metabolism
  • Xenograft Model Antitumor Assays

Substances

  • 3' Untranslated Regions
  • MicroRNAs
  • Nuclear Proteins
  • RNA, Small Interfering
  • TWIST1 protein, human
  • Twist-Related Protein 1
  • Luciferases