WRAP53α基因在顺铂诱导人骨肉瘤U-2OS细胞凋亡中的作用

The role of WRAP53α gene in apoptosis of human osteosarcoma U-2OS cells induced by cisplatin

目的:探讨抑制p53的反义转录本WRAP53α基因表达对顺铂(cisplatin,CDDP)诱导人骨肉瘤U-2OS细胞凋亡的影响。方法:不同浓度的CDDP处理U-2OS细胞后,MTT法检测细胞的增殖率;不同浓度CDDP处理36 h和10μmol/L CDDP处理不同时间以及si WRAP53转染后,实时荧光定量PCR和蛋白质印迹法检测U-2OS细胞中WARAP53α和p53 m RNA及p53蛋白的表达水平;FCM法检测CDDP单独处理及si WRAP53转染后联合CDDP处理组U-2OS细胞的凋亡率。结果:CDDP处理后,U-2OS细胞的增殖率低于未处理的对照组(P<0.05),且呈浓度依赖性。CDDP处理后,U-2OS细胞中WRAP53αm RNA的表达水平明显高于未处理的空白对照组(P<0.05),且呈浓度和时间依赖性;p53 m RNA及p53蛋白的表达水平高于空白对照组(P<0.05)。si WRAP53转染后,WRAP53α和p53 m RNA及p53蛋白的表达水平低于空白对照组(P<0.05)。CDDP单独给药组U-2OS细胞凋亡率高于si WRAP53α转染联合CDDP处理组(P<0.05),2组细胞的凋亡率均高于空白对照组(P<0.05)。结论:CDDP可抑制U-2OS细胞的增殖,诱导WRAP53α和p53 m RNA及p53蛋白的表达;干扰WRAP53α基因的表达可下调p53的表达水平,并逆转CDDP诱导的细胞凋亡。

Objective：To investigate the effect of WRAP53α gene on the apoptosis of human osteosarcoma U-2OS cells induced by cisplatin（CDDP）.Methods：The proliferative rates of U-2OS cells after treatment with different concentrations of CDDP were detected by MTT assay.The expression levels of WRAP53 a and p53 mRNAs as well as p53 protein in U-20 S cells after treatment with different concentrations of CDDP for 36 h or 1 0μmol/L CDDP treatment for different time or transfection with siWRAP53 were measured by realtime fluorescence-based quantitative-PCR and Western blotting,respectively.The apoptotic rates of U-2OS cells after treatment with CDDP alone or transfection with siWRAP53 a in combination with CDDP treatment were determined by flow cytometry（FCM）.Results：The proliferative rate of U-2OS cells after treatment with CDDP was lower than that of the U-2OS cells without any treatment（blank control）（P〈0.05） in a concentration- dependent manner.The expression level of WRAP53 a mRNA in U-2OS cell after treatment with CDDP was significantly higher than that in the blank control group（P〈0.05） in a concentrationand time-dependent manner,while the expression levels of p53 mRNA and p53 protein in U-2OS cell after treatment with CDDP were higher than those in the blank control group（P〈0.05）.The expression levels of WRAP53 a and p53 mRNAs and p53 protein in U-2OS cell after transfection with siWRAP53 a were lower than those in the blank control group（P〈0.05）.The apoptosis rate of U-2OS cells after treatment with CDDP alone was higher than that in the cells transfection with siWRAP53 a and treatment with CDDP（P〈0.05）,and the apoptosis rates of the two groups were both higher than that of the blank control group（P〈0.05）.Conclusion：CDDP can suppress the proliferation of U-2OS cells and induce the expressions of WRAP53 a and p53 mRNAs as well as p53 protein in U-2OS cells.Knockdown of WRAP53αgene can down-regulate the expression level of p53,and reverse the apoptosis of U-20 cells induced by CDDP.