Research Paper Volume 13, Issue 13 pp 17155—17176

Figure 2. EMS sponges miR-758-3p in EC cells, and suppression of miR-758-3p confers the resistance of EC cells to DPP. (A) The expression levels of 8 possible miRNA targets in EC tumor tissues and adjacent normal tissues were determined by RT-qPCR. (B) Sequence comparison of miR-758-3p and wild type or mutated EMS. (C) Dual luciferase assay validated the interaction between EMS and miR-758-3p in ECA-109 cells. (D, E) qPCR determined the miR-758-3p expression levels in ECA-109 cells after EMS knock-down (D) or over-expression (E). (F) qPCR determined the miR-758-3p expression levels in indicated esophageal cancer cell lines and normal cell line HET1A. (G) qPCR results demonstrate that miR-758-3p expression level was significantly decreased in hypoxic ECA-109 cells, in comparison to that of normoxic ECA-109 cells. (H, I) The proliferation of ECA-109 cells after control transfection or transfection of miR-758-3p mimic (H) or miR-758-3p inhibitor (I) and in response to DDP at the indicated concentrations was determined by CCK-8 assays. (J–M) Overexpression of miR-758-3p mediated by mimic transfection in ECA-109 cells significantly increased hypoxic EC cell apoptosis rates in response to DDP, as revealed by Annexin -V flow staining (K); and decreased BCL2 level and increased cleaved caspase-3 level, as revealed by western blot analysis (L). Downregulation of miR-338-5p mediated by inhibitor transfection in ECA-109 cells significantly decreased normoxic EC cell apoptosis under DDP treatment, as revealed by Annexin V flow staining (J), and increased BCL2 level and decreased cleaved caspase-3 level, as revealed by western blot analysis (M). Representative band images from 5 independent experiments with similar results are shown, and n=5 for each group.