Abstract 4366: MicroRNA (miR) 199a-5p regulates mitogen-activated protein kinase 3-11 (MAP3K11) expression in esophageal cancer cells by modulating mRNA stability

Introduction: The MAP kinase signaling pathway regulates several targets involved in proliferation, migration, and apoptosis. MAP3K11 is a critical component of this pathway, whose overexpression has been demonstrated in multiple malignancies. To date, scant information is available on its expression or regulation in esophageal cancer cells. MiR 199a-5p has been shown to post-transcriptionally regulate targets involved with proliferation and apoptosis. Based on sequence analysis, miR 199a-5p is predicted to interact with MAP3K11 with high binding affinity. We hypothesize that miR199a-5p plays a clinically relevant role in regulating proliferative and metastatic properties of esophageal cancer cells through regulation of MAP3K11. The purpose of this study is to measure expression of MAP3K11 and miR-199a-5p in esophageal cancer cells and determine whether miR-199a-5p directly interacts with MAP3K11 mRNA. Methods: Studies were conducted in human esophageal epithelial (hESO) cells and in TE7 and TE10 human esophageal carcinoma cells. Global microRNA expression in these cell lines was determined by microarray analysis and confirmed by real-time PCR. Levels of protein expression were measured by Western blot. MiR-199a-5p function was tested through its overexpression and silencing. mRNA levels were determined using real-time PCR. MAP3K11 mRNA stability was determined by measuring half-life after exposure to Actinomycin D. Binding of miR-199a-5p to MAP3K11 mRNA was examined using a biotinylated RNA pull-down assay. Results: Levels of miR-199a-5p in TE7 and TE10 esophageal cancer cells are reduced by approximately 4,000 log-fold, as compared to hESO cells. MAP3K11 protein expression is markedly elevated in TE7 and TE10 cells compared to hESO cells. MAP3K11 mRNA and protein expression levels decreased in a time-dependent manner following miR-199a-5p overexpression in TE7 and TE10 cells. In reciprocal experiments, silencing miR-199a-5p in hESO resulted in increased MAP3K11 mRNA and protein levels. MAP3K11 mRNA stability is also reduced by ∼ 75% following overexpression of miR-199a-5p in TE7 cells. Binding of miR-199a-5p to MAP3K11 mRNA was confirmed by biotinylated RNA-pull down assay. Conclusions: MiR-199a-5p expression is significantly reduced in TE7 and TE10 esophageal cancer cells relative to hESO cells, while MAP3K11 is markedly over-expressed in TE7 and TE10 cells. MiR-199a-5p directly binds MAP3K11 mRNA and leads to its destabilization. Overexpression of miR-199a-5p in TE7 and TE10 cells results in decreased expression of MAP3K11. Taken together, these results support the supposition that the loss of miR-199a-5p in esophageal epithelial cells contributes to the esophageal oncogenesis. Citation Format: Kimberly A. Byrnes, Pornima Phatak, Daniel Mansour, Jaladanki N. Rao, Douglas Turner, Jian-Ying Wang, James M. Donahue. MicroRNA (miR) 199a-5p regulates mitogen-activated protein kinase 3-11 (MAP3K11) expression in esophageal cancer cells by modulating mRNA stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4366. doi:10.1158/1538-7445.AM2014-4366