慢病毒介导的AP-1基因沉默对卵巢癌SKOV3细胞增殖和凋亡及Survivin表达影响的研究

Effects of lentivirus-mediated AP-1 gene silencing on proliferation,apoptosis and survivin expression of SKOV3 cells

目的探讨慢病毒介导的AP-1基因沉默对卵巢癌SKOV3细胞增殖、凋亡以及Survivin表达的影响。方法基于RNA干扰技术构建慢病毒载体AP-1-shRNA,测定病毒滴度。实时荧光定量PCR及Western blot验证并筛选AP-1-shRNA沉默AP-1效果。将沉默效果最佳的载有AP-1-shRNA的慢病毒感染SKOV3细胞作为实验组,未感染病毒及感染空载体病毒SKOV3细胞作为对照组,用MTT法及Annexin V-APC观察SKOV3细胞增殖、凋亡情况,并用实时荧光定量PCR、Western blot检测SKOV3细胞Survivin表达。结果经测序证实,成功构建AP-1-shRNA-1/2/3三组慢病毒,病毒滴度均大于2×10^8 TU/mL。实时荧光定量PCR及Western blot验证三组病毒均能在mRNA及蛋白水平沉默AP-1的表达,其中AP-1-shRNA-3沉默效果最佳,用于后续实验。沉默AP-1后,SKOV3细胞增殖受到抑制、凋亡增加、Survivin mRNA转录水平和蛋白表达水平降低。结论慢病毒介导的AP-1基因沉默可致卵巢癌SKOV3细胞增殖抑制、凋亡增加,其机制可能与Survivin的下调有关。

Objective To investigate the effects of lentivirus-mediated AP-1 gene silencing on the proliferation,apoptosis and survivin expression of ovarian cancer SKOV3 cells.Methods The lentiviral vector AP-1-shRNA was constructed based on RNA interference technology and the viral titer was determined.RT-PCR and western blotting were used to verify and screen the AP-1-shRNA silencing effects.SKOV3 cells were infected with lentivirus containing AP-1-shRNA,which had the best silencing effect as the experimental group and SKOV3 cells infected with empty vector virus as the control group.The proliferation and apoptosis of SKOV3 cells were observed by MTT and Annexin v-APC,and the expression of survivin in SKOV3 cells was detected by real-time fluorescent quantitative PCR and western blot assay.Results The results of sequencing showed that AP-1-shRNA-1/2/3 lentivirus groups were successfully constructed,and the virus titer was more than 2×10^8 TU/mL.RT-PCR and western blotting confirmed that AP-1 expression was silenced at mRNA and protein levels in AP-1-shRNA-1/2/3 lentivirus groups,in which AP-1-shRNA-3 had the best silencing effects.After AP-1 silencing,SKOV3 cell proliferation was inhibited and apoptosis was increased and the expression of survivin mRNA and protein was decreased.Conclusion Lentivirus-mediated AP-1 gene silencing could inhibit the proliferation and increase the apoptosis of ovarian cancer SKOV3 cells.The mechanism might be related to the down-regulation of survivin.