MicroRNA-200b suppresses arsenic-transformed cell migration by targeting protein kinase Cα and Wnt5b-protein kinase Cα positive feedback loop and inhibiting Rac1 activation

J Biol Chem. 2014 Jun 27;289(26):18373-86. doi: 10.1074/jbc.M114.554246. Epub 2014 May 19.

Abstract

MicroRNA-200b (miR-200b) is a member of miR-200 family that has been found to inhibit cell migration and cancer metastasis; however, the underlying mechanism is not well understood. We previously reported that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells with highly migratory and invasive characteristics, whereas stably re-expressing miR-200b strongly suppresses arsenic-transformed cell migration. This study was performed to investigate how miR-200b inhibits arsenic-transformed cell migration. We found that protein kinase Cα (PKCα) is significantly up-regulated in arsenic-transformed cells. Combining bioinformatics analysis with PKCα 3'-untranslated region vector luciferase reporter assays, we showed that PKCα is a direct target of miR-200b. Inhibiting PKCα activity or knocking down PKCα expression drastically reduced cell migration, phenocoping the inhibitory effect of overexpressing miR-200b. In contrast, forced expression of PKCα in miR-200b overexpressing cells impaired the inhibitory effect of miR-200b on cell migration. In addition, we also found a positive feedback loop between Wnt5b and PKCα in arsenic-transformed cells. Knocking down Wnt5b expression reduced phospho-PKC levels and cell migration; and knocking down PKCα expression decreased Wnt5b level and cell migration. Moreover, forced expression of PKCα increased Wnt5b and phospho-PKC levels and cell migration. Further mechanistic studies revealed that Rac1 is highly activated in arsenic-transformed cells and stably expressing miR-200b abolishes Rac1 activation changing actin cytoskeleton organization. Manipulating PKCα or Wnt5b expression levels significantly altered the level of active Rac1. Together, these findings indicate that miR-200b suppresses arsenic-transformed cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop and subsequently inhibiting Rac1 activation.

Keywords: Cell Migration; Phosphorylation; Rho GTPases; Signal Transduction; Wnt Pathway; microRNA (miRNA).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Arsenic / toxicity*
  • Cell Line, Tumor
  • Cell Movement*
  • Down-Regulation
  • Enzyme Activation
  • Feedback, Physiological
  • Humans
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Protein Binding
  • Protein Kinase C-alpha / genetics*
  • Protein Kinase C-alpha / metabolism
  • Wnt Proteins / genetics*
  • Wnt Proteins / metabolism
  • rac1 GTP-Binding Protein / genetics
  • rac1 GTP-Binding Protein / metabolism*

Substances

  • MIRN200 microRNA, human
  • MicroRNAs
  • WNT5B protein, human
  • Wnt Proteins
  • Protein Kinase C-alpha
  • rac1 GTP-Binding Protein
  • Arsenic