Abstract B227: Overexpression of microRNA 214-3p in esophageal cancer cells enhances sensitivity to chemotherapy-induced apoptosis by targeting CUG-binding protein 1 (CUGBP1) and survivin.

Introduction: Survivin, a key regulator of apoptosis and cell division is overexpressed in esophageal cancer cells and has been correlated with decreased survival. Our previous studies indicate that the RNA-binding protein CUGBP1 is also overexpressed in esophageal cancer cells and contributes to survivin overexpression by stabilizing its mRNA. To date, little information exists on the regulation of CUGBP1 in esophageal cancer cells. Based on sequence analysis, miR 214-3p is predicted to bind both CUGBP1 and survivin mRNA with high binding affinity. The purposes of this study are to determine expression levels of miR 214-3p in esophageal cancer cells and to establish whether miR 214-3p directly interacts with CUGBP1 and survivin mRNA to regulate their expression and modulate sensitivity to chemotherapy-induced apoptosis. Methods: Studies were conducted in human esophageal epithelial (hESO) cells and in TE7 and TE10 human esophageal cancer cells. Global microRNA expression in these cell lines was determined by microarray analysis and confirmed by real-time PCR. MiR 214-3p function was tested by its overexpression and silencing. Levels of protein expression were measured by Western blot. Levels of mRNA were measured by real-time PCR. Binding of miR 214-3p to CUGBP1 and survivin mRNA was examined by biotinylated RNA pull-down assays. mRNA stability was determined by measuring its half-life after addition of Actinomycin D. The apoptotic phenotype was examined by caspase expression using Western blot and flow cytometry. Results: Levels of miR 214-3p in TE7 and TE10 esophageal cancer cells are reduced by approximately 3,000 log-fold in comparison to hESO cells. Biotinylated RNA-pull down assays confirm direct binding of miR 214-3p to CUGBP1 and survivin mRNA. CUGBP1 and survivin mRNA and protein levels are reduced in a time-dependent manner following miR 214-3p overexpression in TE7 and TE10 cells. Conversely, silencing miR 214-3p in hESO cells results in increased CUGBP1 and survivin mRNA and protein levels. The stability of both CUGBP1 and survivin mRNA is decreased following overexpression of miR 214-3p in TE7 cells. Caspase-3 protein levels are increased when TE7 cells are exposed to camptothecin following microRNA 214-3p overexpression. Conclusion: MiR 214-3p expression is markedly reduced in TE7 and TE10 esophageal cancer cells relative to hESO cells. MiR 214-3p binds and destabilizes both CUGBP1 and survivin mRNA. Overexpression of miR 214-3p in esophageal cancer cells results in decreased expression of CUGBP1 and survivin and leads to increased sensitivity to chemotherapy-induced apoptosis. These results strongly support the supposition that the loss of miR 214-3p in esophageal cancer cells contributes to enhanced resistance to chemotherapy-induced apoptosis by leading to increased CUGBP1 and survivin expression. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B227. Citation Format: Pornima Phatak, Daniel Mansour, Kimberly Byrnes, Shan Cao, Lan Liu, Ruiyun Li, Rao Jaladanki, Douglas J. Tuner, Jian Ying Wang, James M. Donahue. Overexpression of microRNA 214-3p in esophageal cancer cells enhances sensitivity to chemotherapy-induced apoptosis by targeting CUG-binding protein 1 (CUGBP1) and survivin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B227.