MicroRNA-200b suppresses arsenic-transformed cell migration by targeting protein kinase Cα and wnt5b-protein kinase Cα positive feedback loop and inhibiting Rac1 activation

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 arsenictransformed cell migration. This study was performed to investigate how miR-200b inhibits arsenic-transformed cell migration. We found that protein kinase Calpha; (PKCalpha;) is significantly up-regulated in arsenic-transformed cells. Combining bioinformatics analysis with PKCalpha; 3alpha;-untranslated region vector luciferase reporter assays, we showed that PKCalpha; is a direct target of miR-200b. Inhibiting PKCalpha; activity or knocking down PKCalpha; expression drastically reduced cell migration, phenocoping the inhibitory effect of overexpressing miR-200b. In contrast, forced expression of PKCalpha; 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 PKCalpha; in arsenic-transformed cells. Knocking down Wnt5b expression reduced phospho-PKC levels and cell migration; and knocking down PKCalpha; expression decreased Wnt5b level and cell migration. Moreover, forced expression of PKCalpha; 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 PKCalpha; 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 PKCalpha; and Wnt5b-PKCα positive feedback loop and subsequently inhibiting Rac1 activation.