PEBP4通过对p53的调节作用降低A549细胞对DDP诱导的细胞毒性

Down regulation of PEBP4 sensitizes drug-resistant lung cancer cells

目的检测肺癌细胞系A549中PEBP4的异位表达与顺铂（DDP）诱导的细胞毒性之间的关系，并探讨其相关作用机制。方法构建pcDNA3-PEBP4重组质粒，转染肺癌细胞A549，检测各组A549细胞中PEBP4的表达。MTF分别检测转染A549细胞，并在DDP作用48h后，各组A549细胞活力的变化；检测各组A549细胞凋亡的变化和p53蛋白的表达变化。应用荧光素酶报告基因系统验证PEBP4是miR-34a的靶基因，并采用蛋白印迹法检测miR-34a对A549细胞中PEBP4蛋白表达的影响。结果pcDNA3-PEBP4转染组PEBP4蛋白的表达显著高于正常对照组和PEBP4shRNA转染对照组（P〈0．01）。DDP作用48h后，DDP加药组的A549细胞活力显著低于正常对照组（P〈0．01）；pcDNA3-PEBP4转染组A549细胞活力低于正常对照组（P〈0．05）；而PEBP4shRNA转染组细胞活力则显著低于正常对照组（P〈0．01）和DDP加药组（P〈0．05）。流式细胞仪检测结果和蛋白印迹法检测结果显示：DDP加药组的凋亡细胞数和p53蛋白的表达水平显著高于正常对照组（P〈0．01）；pcDNA3-PEBP4转染组凋亡细胞数和p53的表达水平高于正常对照组（P〈0．05），却低于DDP加药组和PEBP4shRNA转染组（P〈0．05）；而PEBP4shRNA转染组凋亡细胞数和p53的表达水平则显著高于正常对照组（P〈0．01）和DDP加药组（P〈0．05）。荧光素酶报告基因系统检测结果显示：与对照组相比，转染miR-34amimic组的相对荧光素酶活力显著降低（P〈0．01）。在A549细胞中转染miR-34amimic48h后，细胞中PEBP4蛋白的表达显著下降（P〈0．01）。结论PEBP4的过表达降低A549细胞对DDP诱导的细胞毒性的敏感性，这一过程主要通过影响p53蛋白的表达或miR-34a的调节来完成。

Objective To detect the relationship between expression of in lung cancer cell line A549 and the cell toxicity induced by cisplatin （DDP） , and to investigate the mechanism of the effect of ectopic expression of PE-binding protein 4 （PEBP4）. Methods Construction of pcDNA3-PEBP4 recombinant plasmid, and then transfected into lung cancer cell A549, detection of A549 cells in each group PEBP4 expression. MTT were detected in transfected A549 cells, and after 48 h of DDP treatment, the changes of A549 cell viability in each group were detected by A549 cell apoptosis and the expression of p53 protein. We employed a luciferase reporter-gene assay to confirm PEBP4 as a target gene of miR-34a and the effect of miR-34a on the expression of PEBP4 in A549 cells was detected by Western blot. Results The expression of PEBP4 protein in pcDNA3-PEBP4 transfected group was significantly higher than that in control group and shRNA PEBP4 transfection group （ P 〈 0.01 ）. After 48 h of DDP treatment, MTT assays indicated that A549 cell viability was significantly lower in the DDPtreated 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 ）. Flow cytometry detection results and Western blot detection results showed that dosing of the DDP-treated group, the apoptotic cell number and p53 protein expression level was significantly higher than the normal control group （P 〈0.01 ）; pcDNA3-PEBP4 transfection group apoptosis cell number and p53 expression levels were higher than the normal control group （P 〈 0.05 ）, lower than that of DDP plus drug group and PEBP4 shRNA transfected group （P 〈 0.05 ） ; and PEBP4 shRNA transfeeted group apoptosis and p53 expression level was significantly higher than that of normal control group （P 〈 0.01 ） and DDP and medication group （P 〈 0.05 ）. 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 ）. In A549 cells transfeeted with mimic miR-34a 48 h, the expression of PEBP4 protein was significantly decreased（ P 〈 0.01 ）. Conclusion Overexpression of PEBP4 reduced the sensitivity of A549 cells to DDP induced cytotoxicity, which was mainly accomplished by affecting the expression of p53 protein or by the regulation of miR-34a.