Introduction: Twist1, a basic helix‐loop‐helix (bHLH) transcription factor, has an important role in tumor biology by regulating epithelial‐mesenchymal transition. It also functions as a transcription repressor in NFκB‐dependent cytokine expression. Recently, we reported the identification and characterization of epithelial ovarian cancer (EOC) cells with stem‐like properties (Type I EOC cells). At the microRNA level, Type I EOC cells are characterized by low levels of hsa‐miR‐199a2 and hsa‐miR‐214. We also demonstrated the capacity of Type I EOC cells to differentiate into mature ovarian cancer cells (Type II EOC cells), which has lost stemness potential and express high levels of both hsa‐miR‐199a2 and hsa‐miR‐214. We also showed previously that hsa‐miR‐199a2 is able to regulate the levels of IKKβ and therefore have a direct effect on the NFκB pathway. Still, another group reported the regulatory control of hsa‐miR‐214 on the Akt pathway. In this study, we show that Twist1 is able to regulate the miR‐199a2/214 cluster in EOC cells and can therefore control both NFκB and Akt pathways.
Methods: 5′‐RACE and 3′‐RACE was used to clone the full length pre‐miR‐199a gene. Real‐time PCR was used to determine the levels of Twist1 and miR‐199a2/214. Twist1 was knocked‐down in Type II EOC cells using siRNA. The effect of Twist1 knockdown on levels of IKKβ was determined by Western blot analysis. Cytokines were quantified using Luminex technology.
Results: Characterization of full length of pre‐miR‐199a2 transcript reveals that the MIR199A2 gene contains a human microRNA cluster, miR‐199a2/214, and pre‐miR‐199a2 within the human Dnm3os gene (NCBI GeneID 474332). PCR analysis showed low levels of Twist1 and miR‐199a2/214 in Type I EOC cells but high levels of expression in Type II EOC cells. Knockdown of Twist1 in Type II cells induce a significant decrease in the levels both hsa‐miR‐199a2 and hsa‐miR‐214, and significant increase in IKKβ expression. Ectopic expression of hsa‐miR‐199a2 partially reverse the effect of Twist‐1 knockdown on the levels of IKKβ. The combination of knockdown Twist1 and TNFα stimulation in Type II EOC cells significantly increase Rantes, an NFκB‐dependent cytokine.
Conclusion: We demonstrate for the first time that Twist1 plays a feedback role for the NFkB pathway by repressing IKKβ expression through hsa‐miR‐199a2. Twist1 inhibition of IKKβ expression through regulating hsa‐miR‐199a2, represents a novel negative feedback loop for the NFκB pathway. The demonstration that Twist1 can regulate the miR‐199a2/214 cluster and therefore both the IKKβ and Akt survival pathways, suggests the potential role of Twist1 in EOC differentiation. Furthermore it opens the opportunity to develop new approaches for targeting the ovarian cancer stem cells.
Citation Information: Cancer Res 2009;69(23 Suppl):B75.
- ©2009 American Association for Cancer Research