Downregulation of PEBP4, a target of miR-34a, sensitizes drug-resistant lung cancer cells.

Abstract

The aim of this study was to determine the relationship and underlying mechanisms between ectopic expression of phosphatidylethanolamine-binding protein 4 (PEBP4) and cisplatin (DDP)-induced cytotoxicity in the lung cancer cell line A549 to provide an experimental basis for future chemotherapeutic applications involving PEBP4 in human lung cancer. A recombinant plasmid, pcDNA3-PEBP4, and a PEBP4-targeting small hairpin RNA (shRNA) were transfected into the lung cancer cell line A549. The PEBP4 protein expression levels were determined for each group by Western blot, and after 48 h of cisplatin (DDP) treatment, the viability of cells in the treatment and control groups was determined by 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis in each treatment group was determined using flow cytometry. Western blotting was used to examine expression of the p53 protein in A549 cells from each group. We employed a luciferase reporter-gene assay to confirm PEBP4 as a target gene of miR-34a. Western blotting was used to determine the effects of miR-34a on PEBP4 protein expression in A549 cells. Following transfection of A549 cells with either the recombinant plasmid pcDNA3-PEBP4 or a PEBP4-targeting shRNA, Western blotting analyses showed PEBP4 protein expression was significantly higher in the pcDNA3-PEBP4-transfected group compared with the control or PEBP4-shRNA-transfected groups (p < 0.01). Furthermore, PEBP4 protein expression was significantly reduced in the PEBP4-shRNA-transfected group (p < 0.01). After 48 h of DDP treatment, MTT assays indicated that A549 cell viability was significantly lower in the DDP-treated group compared with the control group (p < 0.01). The viability of A549 cells in the pcDNA3-PEBP4-transfected group was lower than that in the control group (p < 0.05) but higher than that in either the DDP-treated or PEBP4-shRNA-transfected groups (p < 0.05). Moreover, the viability of A549 cells in the PEBP4-shRNA-transfected group was significantly lower than that in either the control (p < 0.01) or DDP-treated (p < 0.05) groups. Flow cytometry and Western blotting analyses indicated that the number of apoptotic cells and p53 protein expression were significantly higher in the DDP-treated group compared with the control group (p < 0.01). In the pcDNA3-PEBP4-transfected group, the number of apoptotic cells and p53 protein expression level were higher than those in the control group (p < 0.05) but lower than those in the DDP-treated and PEBP4-shRNA-transfected groups (p < 0.05). The number of apoptotic cells and p53 protein expression level in the PEBP4-shRNA-transfected group were higher than those in the control (p < 0.01) and DDP-treated (p < 0.05) groups. The luciferase reporter-gene assay showed that the relative luciferase activity after transfection with a miR-34a mimic was significantly reduced compared with the control group (p < 0.01). Western blotting analysis demonstrated that PEBP4 protein expression was significantly decreased in A549 cells 48 h after transfection with a miR-34a mimic compared with the control group (p < 0.01). In conclusion, overexpression of PEBP4 reduced the sensitivity of A549 cells to DDP-induced cytotoxicity, mainly through the altered expression of the p53 protein or the modulation of miR-34a.