Abstract B089: MicroRNA (miR) 214-3p targets ras-related protein14 (RAB14) to inhibit cell migration and invasion in esophageal cancer cells

Objectives: MiR-214-3p acts as a tumor suppressor in various malignancies. We have previously shown that miR-214-3p is markedly downregulated in esophageal cancer cell lines compared to esophageal epithelial cells and its downregulation contributes to chemoresistance in esophageal cancer cells. In several miR-target sequence analysis programs, miR-214-3p is predicted to bind RAB14 mRNA with high affinity. RAB14 is a member of the ras-associated binding protein family of low molecular mass GTPases that are involved in membrane trafficking. Its role in cancer is not well known. The objective of this study was to determine expression of RAB14 in esophageal cancer cells as well as to investigate the interaction between miR-214-3p and RAB14 in these cells and to characterize the functional implications of this interaction. Methods: Studies were performed in human esophageal epithelial (hESO) cells and in FLO1, SKGT4, and TE7 human esophageal cancer cells. Expression of miR-214-3p and RAB14 mRNA in these cell lines was measured by real-time PCR. RAB14 protein expression levels were examined by Western blot. Function of miR-214-3p was tested through its overexpression and silencing. Association of miR-214-3p with RAB14 mRNA was established using a biotinylated RNA pull-down assay and was confirmed by luciferase reporter assay. Cellular proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. xCELLigence RTCA system was used to access cell migration and invasion in real time. Results: Levels of miR-214-3p in FLO1, SKGT4, and TE7 esophageal cancer cells are markedly reduced compared to hESO cells. RAB14 mRNA and protein expression are correspondingly elevated in these cells compared to hESO cells. RAB14 mRNA and protein expression levels decreased in a time-dependent manner following miR-214-3p overexpression in FLO1, SKGT4, and TE7 cells. In reciprocal experiments, silencing miR-214-3p in hESO cells resulted in increased RAB14 mRNA and protein levels. Direct interaction of miR-214-3p and RAB14 mRNA was confirmed by biotinylated RNA-pull down assay and luciferase reporter construct. The stability of RAB14 mRNA is decreased following ectopic expression of miR-214-3p in FLO1 cells. Forced expression of miR-214-3p in FLO1 and SKGT4 cells led to a marked decrease in cellular proliferation, migration, and invasion. Conclusions: MiR-214-3p expression is significantly reduced in FLO1, SKGT4, and TE7 esophageal cancer cells relative to hESO cells. MiR-214-3p directly binds and destabilizes RAB14 mRNA. Forced expression of miR-214-3p results in significantly decreased RAB14 mRNA and protein expression, leading to decreased cellular proliferation, migration, and invasion in esophageal cancer cells. These results add to the roles by which miR-214-3p functions as a tumor suppressor in esophageal cancer cells. Citation Format: Pornima Phatak, Jaladanki N. Rao, Carol B. Fowler, Douglas J. Turner, Jian-Ying Wang, James M. Donahue. MicroRNA (miR) 214-3p targets ras-related protein14 (RAB14) to inhibit cell migration and invasion in esophageal cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B089.