摘要
目的探讨neu RNAi对卵巢癌耐药细胞株SKOV3/DDP细胞生长、凋亡及药物敏感性的作用,寻找下调基因表达并逆转化疗耐药的方法 ,为卵巢癌的基因治疗探索新途径。方法应用含有U6启动子的pSilencer 1.0-U6表达载体构建neu基因的小干扰RNA(siRNA)表达载体(pSilencer-neu),用脂质体方法将pSilencer-neu转染卵巢癌顺铂耐药细胞株SKOV3/DDP,另设未转染组及转染pSilencer-control组为对照。显微镜下观察转染前后细胞的变化;用RT-PCR及Western Blot检测neu基因mRNA及蛋白的表达;MTT法检测细胞生长情况;流式细胞仪检测细胞凋亡及周期的变化;加入顺铂后检测RNAi对SKOV3/DDP顺铂药物敏感性的影响。结果 neu RNAi可明显下调卵巢癌耐药细胞株SKOV3/DDP中neu的基因表达;使其细胞生长受到抑制,生长速度减慢,生长曲线低平;细胞凋亡增加且细胞周期发生变化,G_1/G_0期细胞增多,S期细胞则减少;顺铂耐药实验显示,转染RNAi的SKOV3/DDP细胞IC_(50)明显下调,细胞凋亡率显著增加。结论应用RNAi可以部分阻抑neu基因在卵巢癌顺铂耐药细胞株SKOV3/DDP中的表达,使SKOV3/DDP细胞生长减慢、凋亡增加,而且对顺铂的敏感性增强。
Objective To explore the effects of RNA interference （RNAi） on the growth, apoptosis, and chemotherapeutic sensitivity of ovarian cancer cell line SKOV3/DDP. Methods Expression vector of Neu gene - targeting siRNA was constructed using pSileneer 1.0 -U6 vector contained U6 promotor （pSilencer- neu） and transfeeted into SKOV3/DDP cells by lipofeetamine. Untransfected group and pSilencer - control group were used as controls. The expression of Neu mRNA and protein were identified by RT - PCR and Western blot. The proliferation of SKOV3/DDP cells was examined by MTT assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. Cisplatin （DDP） resistant experiment was performed on SKOV3/DDP cells with siRNA. Results Neu RNAi plasmid knockeddown Neu expression in SK- OV3/DDP cells obviously, arrested the cells in G1/G0 phase, inhibited the cell proliferation, and promoted cell apoptosis. The IC50 of DDP for SKOV3/DDP cells transfected with Neu siRNA decreased. Conclusion Neu RNAi can partly suppress the expression of Neu in SKOV3/DDP cells, inhibit the cell proliferation, and promote cell apoptosis. Neu RNAi can enhance the cell sensitivity to apoptosis induced by cisplatin, which implies that Neu RNAi may partly reverse the drug resistance of ovarian cancer. RNAi is potentially a new approach for gene therapy of cancer.
出处
《癌症进展》
2010年第4期394-400,共7页
Oncology Progress
基金
国家自然科学基金资助(项目编号:30271470)
