E3泛素连接酶对卵巢癌细胞系SKOV3/DDP顺铂耐药性的影响

Effect of E3 isolated by differential display on Cisplatin resistance of SKOV3/DDP cells

目的探讨E3泛素连接酶(EDD)基因在SKOV3/DDP细胞顺铂耐药中的作用。方法采用RT-PCR和Western blot检测人卵巢癌SKOV3细胞和SKOV3/DDP细胞内EDD表达水平的差异。通过转染EDD-si RNA沉默EDD的表达后,噻唑蓝法检测顺铂对SKOV3/DDP细胞增殖的影响,Annexin V-FITC/PI双染法检测顺铂对细胞凋亡的影响;Western blotting检测髓样细胞白血病-1(Mcl-1)和细胞周期检测点激酶2(CHK2)蛋白表达水平。结果SKOV3细胞EDD的表达水平显著低于卵巢癌耐药细胞SKOV3/DDP(P<0.05)。EDD-si RNA转染24 h后,EDD m RNA和蛋白表达水平均显著低于未转染组(P<0.05);SKOV3/DDP细胞IC50值显著降低(P<0.05),Mcl-1蛋白的表达受到抑制(P<0.05),而CHK2无明显变化(P>0.05)。结论 EDD-si RNA能沉默EDD基因,降低Mcl-1蛋白的表达水平,增强顺铂对SKOV3/DDP细胞的促凋亡作用,降低SKOV3/DDP细胞对顺铂的耐药性,有效恢复SKOV3/DDP细胞顺铂敏感性。

Objective To discuss the effect of E3 isolated by differential Display （EDD） on Cisplatin resistance of SKOV3/DDP cells. Methods The expression difference of EDD between Human ovarian cancer cells SKOV3 and SKOV3/DDP cells was detected by Western blot and RT-PCR; EDD-siRNA was transfected into SKOV3/DDP cells to silence the expression of EDD, MTT assay was used to detect the chemosensitivity of SKOV3/DDP cells to Cisplatin, the apoptosis induced by Cisplatin was tested by Annexin V-FITC/PI, the protein levels of Mcl-1 and CHK2 was measured by Western blotting. Results The expression of EDD in SKOV3 cells was significantly lower than SKOV3/EDD cells （P〈0.05）. EDD expression was successful silenced and the IC50 of SKOV3/DDP cells was significantly decreased at 24 h in EDD-siRNA group compared with the control group （P〈0.05）. Conclusion Silencing EDD gene with EDD-siRNA reduce the expression of Mcl-1, enhance the apoptosis in-ducing effect of Cisplatin on SKOV3/DDP cells, depress the Cisplatin resistance of SKOV3/DDP cells and effectively recover SKOV3/DDP cell sensitivity to Cisplatin.