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 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.
From the Department of Physiology, Michigan State University, East Lansing, Michigan 48824.
Zhishan
Z
Wang
From the Department of Physiology, Michigan State University, East Lansing, Michigan 48824.
Brock
B
Humphries
From the Department of Physiology, Michigan State University, East Lansing, Michigan 48824.
Hua
H
Xiao
the Institute for Chemical Carcinogenesis, State Key Laboratory of Respiratory Diseases, Guangzhou Medical University, Guangzhou 510182, China, and.
Yiguo
Y
Jiang
From the Department of Physiology, Michigan State University, East Lansing, Michigan 48824, the Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824 yangcf@msu.edu.
Chengfeng
C
Yang
D001151:Arsenic / Q000633:toxicity*; D045744:Cell Line, Tumor; D002465:Cell Movement; D015536:Down-Regulation; D004789:Enzyme Activation; D025461:Feedback, Physiological; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000235:genetics* / Q000378:metabolism; D051153:Wnt Proteins / Q000235:genetics* / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
C533896:MIRN200 microRNA, human; D035683:MicroRNAs; C435831:WNT5B protein, human; D051153:Wnt Proteins; D051571:Protein Kinase C-alpha; D020830:rac1 GTP-Binding Protein; D001151:Arsenic
Cell Migration; Phosphorylation; Rho GTPases; Signal Transduction; Wnt Pathway; microRNA (miRNA)