目的:探究沉默多聚腺苷二磷酸核糖聚合酶1(PARP-1)对乳腺癌细胞MCF-7顺铂耐药的影响及可能的作用机制.方法:Western blot及real-time PCR实验检测乳腺癌细胞株MCF-7及相应耐药细胞株MCF-7/DDP中PARP-1的表达情况.采用转染PARP-1小干扰RNA(siRNA)的方法沉默乳腺癌耐药细胞株MCF-7/DDP中PARP-1的表达,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测PARP-1、Bcl-2、Bax、cleaved caspase-3、caspase-3、细胞色素C(Cyto-C)、细胞外调节蛋白激酶(ERK)和磷酸化ERK(p-ERK)的蛋白水平.结果:耐药细胞株MCF-7/DDP中PARP-1的mRNA及蛋白表达水平均明显高于亲本细胞株(P<0.05);并且在亲本细胞株MCF-7中加入顺铂刺激后,PARP-1的蛋白表达水平明显升高(P<0.05).沉默PARP-1可诱导乳腺癌耐药细胞株MCF-7/DDP的凋亡,增强细胞对顺铂的敏感性,还可下调Bcl-2/Bax 和p-ERK的蛋白水平,同时上调cleaved caspase-3及Cyto-C的蛋白水平.而应用ERK特异性抑制剂U0126后,PARP-1沉默组的细胞活力进一步降低.结论:沉默乳腺癌耐药细胞株MCF-7/DDP中PARP-1的表达可恢复细胞对顺铂的敏感性,促进细胞经线粒体途径的凋亡,其机制可能与其抑制ERK的磷酸化,进而阻断该信号通路有关.%AIM:To study the effect of poly(ADP-ribose)polymerase-1(PARP-1)on cisplatin resistance of human breast cancer MCF-7 cells and its possible mechanisms.METHODS: The expression of PARP-1 at mRNA and protein levels in MCF-7 cells and MCF-7/DDP cells was determined by real-time PCR and Western blot.The expression of PARP-1 in the MCF-7/DDP cells was blocked by PARP-1 siRNA.The cell viability and apoptosis were detected by the CCK-8 assay and flow cytometry analysis,respectively.Furthermore, the protein levels of PARP-1, Bcl-2, Bax, cleaved caspase-3,caspase-3,cytochrome C(Cyto-C),extracellular signal-regulated kinase(ERK)and phosphorylated ERK(p-ERK)were detected by Western blot.RESULTS:The expression of PARP-1 at both mRNA and protein levels was signifi-cantly up-regulated in the MCF-7/DDP cells.The expression of PARP-1 was increased in the MCF-7 cells treated with cis-platin.Knockdown of PARP-1 induced the apoptosis of MCF-7/DDP cells with an increased sensitivity to cisplatin.Mean-while,knockdown of PARP-1 down-regulated the protein levels of Bcl-2/Bax and p-ERK, but up-regulated the protein levels of cleaved caspase-3 and Cyto-C.After incubated with a specific ERK inhibitor U 0126,the cell viability in PARP-1 siRNA group was down-regulated significantly.CONCLUSION:Knockdown of PARP-1 increases the sensitivity of MCF-7/DDP cells to cisplatin,and promotes the cell apoptosis via mitochondrial apoptosis pathway.The mechanism may be re-lated to the attenuation of ERK signaling pathway by inhibiting phosphorylation of ERK.
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