RNA干扰Survivin基因沉默对卵巢癌SKOV3及SKOV3/ADM细胞凋亡的影响

Effect of RNAi-mediated Survivin Gene Silencing on Apoptosis of Ovarian Cancer Cell Lines SKOV3 and SKOV3/ADM

背景与目的:RNA干扰技术被广泛应用于肿瘤基因治疗、抗病毒感染、基因药物筛选等许多领域。Survivin基因在卵巢癌细胞株SKOV3及其耐药株SKOV3/ADM中高表达,是进行卵巢癌基因治疗理想的靶点。本研究探讨Survivin基因沉默后对卵巢癌细胞SKOV3及其耐药株SKOV3/ADM凋亡的影响。方法:将重组质粒pshRNA鄄Survivin以脂质体转染至SKOV3、SKOV3/ADM细胞。分别用半定量RT鄄PCR、Westernblot检测细胞内SurvivinmRNA和蛋白表达的变化,吖啶橙/溴乙锭(AO/EB)染色法、TUNEL及流式细胞仪检测重组质粒诱导细胞凋亡的情况。结果:转染pshRNA鄄Survivin后,SKOV3、SKOV3/ADM细胞中SurvivinmRNA拷贝数明显减少,抑制率分别为83.79%和91.56%,蛋白质的表达水平显著降低;干扰组细胞凋亡率显著高于对照组(P<0.01)。流式细胞仪检测结果显示,Survivin沉默后48h,SKOV3、SKOV3/ADM的细胞凋亡率分别为14.05%和21.02%,而在对照组未检测到明显凋亡。结论:重组质粒pshRNA鄄Survivin可明显抑制SKOV3、SKOV3/ADM细胞内SurvivinmRNA和蛋白的表达,介导卵巢癌细胞凋亡。

BACKGROUND ＆ OBJECTIVE： RNA interference （RNAi） has been widely used in tumor gene therapy, antivirus, and gene drug selection.Survivin gene is highly expressed in ovarian cancer cell line SKOV3 and drugresistant cell line SKOV3/ADM, and is an ideal target of gene therapy for ovarian cancer. This study was to explore effect of RNAi-mediated Survivin gene silencing on apoptosis of SKOV3 and SKOV3/ADM cells. METHODS：Recombinant plasmid pshRNA-Survivin was transfected into SKOV3 and SKOV3/ADM cells. The expression of Survivin was detected by semiquantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The apoptosis of SKOV3 and SKOV3/ADM cells after transfection were evaluated by AO/EB dyeing, TUNEL, and flow cytometry.RESULTS. After transfection of pshRNA-Survivin, mRNA and protein levels of Survivin gene in SKOV3 and SKOV3/ADM cells were obviously reduced; the apoptosis of SKOV3 and SKOV3/ADM cells were significantly higher in transfection group than in control group （P〈 0.01）; 48 h after transfection,the apoptosis rate was 14.05% in SKOV3 cells, and 21.02% in SKOV3/ADM cells, no obvious apoptosis was detected in control cells. CONCLUSION：pshRNA-Survivin could reduce the expression of Survivin gene, and induce apoptosis of SKOV3 and SKOV3/ADM cells.