RNA干扰技术抑制卵巢上皮性癌耐药细胞TopoⅡα基因表达并逆转耐药的体外研究

Inhibition of TopoⅡα gene expression and reversing of drug resistance in multi-drug resistant epithelial ovarian cancer cells induced by RNA interference in vitro

目的探讨RNA干扰技术抑制卵巢上皮性癌（卵巢癌）耐药细胞TopoⅡα基因的表达是否能逆转耐药。方法（1）设计并筛选针对TopoⅡα基因的、沉默效果最佳的小分子干扰RNA（siRNA），将其克隆到psilencer4．1-CMV-neo载体上；将psilencer4．1-CMV—neo—TopoⅡα重组质粒转染至受试细胞，建立稳定转染重组质粒的细胞TopoⅡα siRNA（＋）SKOV3／DDP。（2）采用RT—PCR技术和蛋白印迹法测定稳定转染细胞中TopoⅡαmRNA和蛋白的表达；分别采用四甲基偶氮唑蓝比色法、流式细胞仪和高效液相色谱法测定TopoⅡαRNA干扰前后细胞的耐药指数、细胞周期和细胞内顺铂含量的变化。结果（1）转染psilencer4．1-CMV-neo—TopoⅡα质粒后能抑制SKOV3／DDP细胞中TopoⅡα基因的表达，TopoⅡαsiRNA（＋）SKOV3／DDP和SKOV3／DDP细胞中TopoⅡαmRNA的表达水平分别为0和0．92±0．08；两种细胞中TopoⅡα蛋白的表达水平分别为0．51±0．04和1．95±0．09，两者比较，差异有统计学意义（P〈0．01）。（2）SKOV3／DDP细胞转染TopoⅡαsiRNA后耐药指数下降，TopoⅡαsiRNA（＋）SKOV3／DDP和SKOV3／DDP细胞的耐药指数分别为3．46和5．05，两者比较，差异有统计学意义（P〈0．05）。（3）TopoⅡαsiRNA（＋）SKOV3／DDP细胞的G0／G1期、G2／M期细胞比例较SKOV3／DDP细胞增加，S期细胞比例减少（P均〈0．05）。（4）TopoⅡαsiRNA（＋）SKOV3／DDP细胞经20μg／ml顺铂处理24h后，其顺铂含量（157．20ng）较SKOV3／DDP细胞（63．99ng）升高，两者比较，差异有统计学意义（P〈0．05）。结论阻断卵巢癌耐顺铂细胞中TopoⅡα基因的表达能逆转其对顺铂的耐受性。

Objective To explore whether or not muhi-drug resistance could be reversed by RNA interference the expression of Topo Ⅱα gene in epithelial ovarian cancer cell lines in vitro. Methods （ 1 ） The best silent small interference RNA （siRNA） of Topo Ⅱα gene was designed and chose and cloned into psilencer4. 1-CMV-neo vector. The psilencer4. 1-CMV-neo-Topo Ⅱα was transfected into SKOV3/DDP cell, then Topo Ⅱα siRNA （ ＋ ） SKOV3/DDP ceils was incubated. （ 2 ） The Topo Ⅱα mRNA and protein expression of the stability-transfecting cell lines were detected by RT-PCR and western blot method, respectively. The resistance index, the cell cycle and the cellular content of cisplatin were detected by methyl thiazolyl tetrazolium assay, the flow eytometry and high performance liquid chromatography method before and after Topo Ⅱα RNA interference in cells. Results （ 1 ） The Topo Ⅱα gene expression level in SKOV3/DDP cells could be inhibited after the plasmid DNA psilencer4.1-CMV-neo-Topo Ⅱα transfeced. The expression level of TopoⅡα mRNA in Topo Ⅱα siRNA （ ＋ ） SKOV3/DDP and SKOV3/DDP cells were 0 and 0. 92 ±0. 08 ; the expression level of Topo Ⅱα protein in Topo Ⅱα siRNA （ ＋ ） SKOV3/DDP and SKOV3/DDP ceils were 0. 51±0. 04 and 1.95±0. 09 （P 〈0. 01 ）. （2） The muhi-drug resistance index of TopoⅡα siRNA （ ＋ ） SKOV3/DDP cell was significantly lower compared with that in SKOV3/DDP cell （3.46 vs 5.05, P 〈0. 05）. （3） The percentage of G0/G1 and GE/M phase cell in Topo Ⅱα siRNA（ ＋ ） SKOV3/DDP cells were higher than that in SKOV3/DDP cells （ P 〈 0. 05 ）. （4） The content of cisplatin in Topo Ⅱα siRNA（ ＋ ） SKOV3/DDP cells treated with cisplatin for 24 hours was significantly higher than that in SKOV3/DDP cell （ 157.20 vs 63.99 ng, P 〈 0.05 ）. Conclusion The results showed that the tolerance of cisplatin would be reversed by blocking the Topo Ⅱα genc expression in cisplatin-resistant epithelial ovarian cancer cells.