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 transfe...

MicroRNAs as Regulators of Cisplatin Resistance in Lung Cancer

“…These functions of miR-17, 20a, 20b may be caused at least in part via inhibition of TGF beta signal pathway, because those miRNAs are shown to directly target and repress TGFbeta receptor 2 (TGF beta R2) which is an important component of TGF beta signal pathway [36]. In another report, both miR-15b and miR-34a can regulate EMT and the response of lung adenocarcinoma cells to CDDP both in vitro and in vivo by targeting PEBP4 [37,38]. Also, miR-27a can regulate EMT via direct and functional targeting Raf-1 kinase inhibitory protein (RKIP), which then affects the sensitivity of lung adenocarcinoma cells to CDDP both in vitro and in vivo [39].…”

RBMMMDA: predicting multiple types of disease-microRNA associations

“…Especially, among the top 10 and top 20 prediction list, 90% of them have literature evidences (See Table 2 and Supplementary Table 2 ). In the top 10 potential related miRNAs, mir-34 family (a and b), which functions as tumor-suppressive miRNAs to induce apoptosis and inhibit proliferation in lung cancer cells by directly targeting TGFβR2 and Met, are inactivated by CpG methylation at their promoter region 96 97 98 99 100 101 102 103 ; Also, mir-218, 133a and 143 are tumor-suppressors that play roles in inhibiting tumor cell invasion by targeting the tumorigenesis-related genes in lung cancer, such as N-cadherin, oncogenic receptors and so on 104 105 106 107 108 109 110 111 112 113 . There is also confirmed lung cancer-related miRNAs with the third association type.…”

Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions

“…Second, transfection of interference RNA for PEBP4 into cancer cells inhibits cell proliferation, migration and invasion or induces apoptosis, whereas overexpression of this protein causes opposite changes (17, 19, 23–26). Third, the expression levels of PEBP4 correlates with the sensitivity of cancer cells to chemotherapy or radiotherapy in which the upregulation of PEBP4 confers resistance to these therapies while its downregulation enhances the sensitivity (15, 26, 31, 32). These properties of PEBP4 are consistent with a role in promoting tumor transformation and progression, but are inconsistent with the inhibitory effect on the activation of the Raf-1/MEK/ERK pathway.…”