Abstract A37: Overexpression of microRNA (miR) 199a-3p reduces proliferation through the induction of G2/M arrest in esophageal cancer cells by targeting cyclin D1

Objectives: Downregulation of miR-199a-3p has been demonstrated in several malignancies. A potential role of miR-199a-3p as a tumor suppressor has been postulated based on its ability to regulate targets involved in proliferation, apoptosis, and migration. Using miR array analysis, we have previously shown that miR-199a-3p is one of the most markedly downregulated miRs in esophageal cancer cell lines compared to esophageal epithelial cells. To date, no information exists on the role of miR-199a-3p in esophageal cancer cells. In several miR-target sequence analysis programs, miR-199a-3p is predicted to bind cyclin D1 mRNA with high affinity. The objective of this study was to determine expression of cyclin D1 in esophageal cancer cells and to investigate the interaction between miR-199a-3p and cyclin D1 in these cells and to characterize the functional implications of this interaction. Methods: Studies were conducted in human esophageal epithelial (hESO) cells and in TE7 human squamous esophageal cancer cells. Levels of cyclin D1 protein expression were assessed by Western blot. Expression of miR-199a-3p and cyclin D1 mRNA in these cell lines was measured by real-time PCR. MiR-199a-3p function was tested through its overexpression and silencing. Binding of miR-199a-3p to cyclin D1 mRNA was examined using a biotinylated RNA pull-down assay. Cellular proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle progression was determined by FACS analysis. Results: Levels of miR-199a-3p in TE7 esophageal cancer cells are reduced by more than 3 log-fold, as compared to hESO cells. Cyclin D1 protein expression is markedly elevated in TE7 cells compared to hESO cells. Cyclin D1 mRNA and protein expression levels decreased in a time-dependent manner following miR-199a-3p overexpression in TE7 cells. In reciprocal experiments, silencing miR-199a-3p in hESO cells resulted in increased cyclin D1 mRNA and protein levels. Binding of miR-199a-3p to cyclin D1 mRNA was confirmed by biotinylated RNA-pull down assay. Forced expression of miR-199a-3p in TE7 cells led to a marked decrease in cellular proliferation resulting from the induction of G2/M arrest. Conclusions: MiR-199a-3p expression is significantly reduced in TE7 esophageal cancer cells relative to hESO cells. MiR-199a-3p directly binds cyclin D1 mRNA, leading to its destabilization and a marked decreased in cyclin D1 protein expression. This interaction results in decreased cellular proliferation through the induction of G2/M arrest. These results suggest that the loss of miR-199a-3p in esophageal epithelial cells may be an important event in esophageal carcinogenesis. Citation Format: Pornima Phatak, Kimberly Byrnes, Jaladanki N. Rao, Douglas J. Turner, Jian Y. Wang, James M. Donahue. Overexpression of microRNA (miR) 199a-3p reduces proliferation through the induction of G2/M arrest in esophageal cancer cells by targeting cyclin D1. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A37.