Gankyrin基因沉默对人卵巢癌SKOV3/CDDP细胞顺铂耐药性的逆转作用和机制

摘要

背景与目的：卵巢癌是常见的妇科肿瘤，抗肿瘤药耐药性的产生是卵巢癌治疗失败的主要原因之一，Gankyrin基因被认为与肿瘤耐药性密切相关，本文探讨了Gankyrin基因沉默对卵巢癌耐顺铂细胞系SKOV3/DDP顺铂耐药性的逆转作用及机制。方法：应用real-time PCR技术考察Gankyrin在SKOV3和SKOV3/DDP细胞中的表达，应用MTS法检测Gankyrin对SKOV3/DDP细胞顺铂耐受性的影响，应用流式细胞术检测肿瘤细胞凋亡和细胞内罗丹明-123(Rhodamine-123，Rh-123)含量的变化，Western blot和real-time PCR技术检测肿瘤细胞耐药相关蛋白MDR1、Caspase-3/8、Survivin和Bcl-2蛋白表达， Western blot法检测p53、NF-κB和PTEN蛋白表达和AKT磷酸化水平。结果：Gankyrin在SKOV3/DDP细胞中表达升高，沉默Gankyrin基因后可增加SKOV3/DDP细胞对顺铂的敏感性。基因沉默前后耐药逆转倍数(resistant factor，RF)为1.81和2.45，肿瘤细胞中Rh-123含量提高了1.73和2.42倍，细胞凋亡率是对照组的2.23倍和4.23倍，耐药相关蛋白MDR1、Survivin和Bcl-2蛋白水平显著下降，MDR1 mRNA表达是对照组的62.8%和21.6%，Survivin mRNA表达是对照组的24.5%和10.3%，Bcl-2 mRNA表达是对照组的47.5%和18.4%，Caspase-3/8、p53和PTEN表达水平上升，AKT磷酸化和NF-κB水平下降。结论：沉默Gankyrin基因可逆转SKOV3/DDP对顺铂的耐药性，可能与抑制药物外排，促进细胞凋亡有关，PTEN/AKT/NF-κB/p53信号通路可能是其中心环节。%Background and purpose: Ovarian cancer is the common gynecological cancer, and the drug resistance of anti-tumor drug was one of major reasons for therapy failure, some studies considered that there is a closed relationship between Gankyrin and drug resistance. In this study, we investigated the effects and mechanisms of Gankyrin silencing on reversing the cisplatin resistance of ovarian cancer drug-resistant SKOV3/DDP cell line. Methods:The expression of Gankyrin in SKOV3 and SKOV3/DDP cells was measured by real-time PCR assay, MTS assay was employed to determine the effect of Gankyrin on SKOV3/DDP sensitivity to cisplatin, apoptosis rate and intracellular concentration of rhodamine-123 (Rh-123) were determined by lfow cytometry, the expression of multi-drugs resistant protein MDR1, Caspase-3/8, Survivin and Bcl-2 were determined by Western blot and real-time PCR. The phosphorylation of AKT and expression of p53, NF-κB and PTEN were analyzed by Western blot assay. Results:The expression of Gankyrin was increased in SKOV3/DDP cells, Gankyrin silencing was able to increase the cisplatin sensitivity of SKOV3/DDP. Before and after gene silencing, the reverse folds (RF) to cisplatin were 1.81 and 2.45, respectively, the intracellular levels of Rh-123 were 1.73 and 2.42 fold, the apoptosis rates were 2.23 and 4.23 fold,the expressions of MDR1, Survivin and Bcl-2 were downregulated, the mRNA expressions of MDR1 were 62.8%and 21.6%, the mRNA expressions of Survivin were 24.5%and 10.3%, the mRNA expressions of Bcl-2 were 47.5%and 18.4%, the levels of Caspase-3/8, p53 and PTEN were elevated, phosphorylation of AKT and expression of NF-kB were downregulated compared with control group. Conclusion:Gankyrin silencing was able to reverse the cisplatin resistance of SKOV3/DDP cells by inhibiting the drug eflfux and promoting cell apoptosis, the PTEN/AKT/NF-κB/p53 may be the key pathway.