1866 The Role of Microrna-424/503-Wee1 Axis in Ovarian Cancer Stem Like Cells

Study Objective We investigated the role of miR-424/503-WEE1 axis in ovarian cancer and its potential utility as a therapeutic target. Design To determine whether miR 424/503-WEE1 axis is associated with the generation of CSCs, we up/down-regulated miR 424/503-WEE1 axis in ovarian cancer sphere cells. Setting This study was performed in Pusan National University Hospital, Busan, Korea Patients or Participants The human epithelial ovarian cancer cell lines OVCAR3, SKOV3, and OVCAR429 were obtained from the Korean Cell Line Bank. Ovarian cancer cells were maintained in MEM (Life Technologies, Inc., Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 100 μg/ml streptomycin in a humidified 5% CO2 incubator. Interventions In order to assess spheroid formation, approximately 5 × 103 cells were suspended in 10 mL of serum-free DMEM-F12, supplemented with 10 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor and plated in an ultra-low attachment plate to prevent adherence. Spheres were counted at 14 days after plating. All experiments were performed in triplicate. Measurements and Main Results Firstly, we discovered that miR-424 and miR-503 are lowly expressed in ovarian cancer stem-like cells (CSCs) compared with non-CSCs. Over-expression of miR-424 and miR-503 resulted in decreased cancer colony formation, migration in ovarian cancer cell lines. Moreover, miR-424 and miR-503 over-expression caused inhibition of spheroid formation and the DNA damage repair pathway, while over-expression of Wee1 caused increased spheroid formation and cell survival in ovarian CSCs. Secondly, we found that the stem cell marker NANOG decreased miR-424 and miR-503 transcription in ovarian CSCs. In addition, miR-424 and miR-503 bind to the 3’ UTR of WEE1 to decrease its expression. Finally, we found that atorvastatin treatment increased miR-424 and miR-503 expression while it significantly decreased NANOG and WEE1 expression in ovarian CSCs. Conclusion Our findings indicate that increased WEE1 is an oncogenic marker for ovarian CSCs and is a potential target for CSC-specific therapy.