Zhang Xin, Li Ning, Wang Yi-hua, Huang Ying, Xu Ning-zhi, Wu Ling-ying
Department of Obstetrics and Gynecology, China Meitan General Hospital, Beijing 100028, China.
Zhonghua Zhong Liu Za Zhi. 2009 Mar;31(3):174-7.
To explore a new approach in gene therapy of ovarian cancer, we used RNAi to inhibit survivin gene expression, and explore the effect of survivin and neu RNAi on growth, apoptosis and chemosensitivity of ovarian cancer cell line SKOV3/DDP cells.
Expression vector of survivin gene-targeting siRNA was constructed using pSilencer 1.0-U6 vector containing U6 promotor (pSilencer-survivin) and transfected into SKOV3/DDP cells by lipofectamine. The untransfected group and pSilencer-control group were used as control. The expressions of survivin mRNA and protein were identified by RT-PCR and Western blot assay. The proliferation of SKOV3/DDP cells was determined by MTT assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. Cisplatin (DDP) resistance experiment was performed in SKOV3/DDP cells with RNAi.
Survivin RNAi plasmid knocked-down survivin expression in SKOV3/DDP cells obviously, arrested the cells at G(1)/G(0) phase, inhibited the cell proliferation and promoted cell apoptosis. The IC(50) of DDP to SKOV3/DDP cells transfected with survivin siRNA was dropped.
Survivin RNAi can partly suppress the expression of survivin in SKOV3/DDP cells, inhibit the cell proliferation and promote cell apoptosis. Survivin RNAi can enhance the cell sensitivity to apoptosis induced by cisplatin, which implies that survivin RNAi may partly reverse the drug resistance of ovarian cancer. RNAi could be a new approach for gene therapy of cancer.
为探索卵巢癌基因治疗的新方法,我们采用RNA干扰技术抑制生存素基因表达,探讨生存素和neu基因RNA干扰对卵巢癌细胞株SKOV3/DDP细胞生长、凋亡及化疗敏感性的影响。
利用含U6启动子的pSilencer 1.0-U6载体构建靶向生存素基因的小干扰RNA(siRNA)表达载体(pSilencer-survivin),并用脂质体转染SKOV3/DDP细胞。未转染组和pSilencer-对照组作为对照。采用逆转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹法检测生存素mRNA和蛋白的表达。采用噻唑蓝(MTT)法检测SKOV3/DDP细胞的增殖情况。采用流式细胞术分析细胞凋亡率及细胞周期分布。对经RNA干扰处理的SKOV3/DDP细胞进行顺铂(DDP)耐药实验。
生存素RNA干扰质粒明显下调SKOV3/DDP细胞中生存素的表达,使细胞阻滞于G(1)/G(0)期,抑制细胞增殖并促进细胞凋亡。转染生存素siRNA的SKOV3/DDP细胞对DDP的半数抑制浓度(IC(50))降低。
生存素RNA干扰可部分抑制SKOV3/DDP细胞中生存素的表达,抑制细胞增殖并促进细胞凋亡。生存素RNA干扰可增强细胞对顺铂诱导凋亡的敏感性,这提示生存素RNA干扰可能部分逆转卵巢癌的耐药性。RNA干扰可能成为癌症基因治疗的一种新方法。