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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 inhibiting the expression of WRAP53α, an antisense transcript of p53, on the apoptosis of human osteosarcoma U-2OS cells induced by cisplatin (CDDP). Methods: The proliferative rate of U-2OS cells treated with different concentrations of CDDP was detected by MTT assay. CDDP treated with different concentrations of CDDP for 36 h and 10 μmol / L CDDP for different time and si WRAP53 transfection, real-time fluorescence quantitative PCR and Western blotting The expression levels of WARAP53α, p53 m RNA and p53 protein in U-2OS cells were detected. The apoptosis rate of U-2OS cells treated with CDDP alone or combined with CDDP was detected by FCM. Results: The proliferation rate of U-2OS cells after CDDP treatment was lower than that of the untreated control group (P <0.05), and in a concentration-dependent manner. After CDDP treatment, the expression level of WRAP53αmRNA in U-2OS cells was significantly higher than that in untreated blank control group (P <0.05), and the expression of p53 m RNA and p53 protein was higher than that of blank control Group (P <0.05). si WRAP53 transfected WRAP53α and p53 m RNA and p53 protein expression levels were lower than the blank control group (P <0.05). The apoptosis rate of U-2OS cells in CDDP alone group was higher than that of si WRAP53α transfection group (P <0.05). The apoptosis rate of two groups was higher than that of blank control group (P <0.05). CONCLUSION: CDDP can inhibit the proliferation of U-2OS cells and induce the expression of WRAP53α, p53 m RNA and p53 protein. Interfering with WRAP53α gene expression can down-regulate the expression of p53 and reverse the apoptosis induced by CDDP.