RNA干扰体外诱导多药耐药上皮性卵巢癌细胞中Topo IIα基因表达抑制及耐药逆转

[Inhibition of Topo II alpha gene expression and reversing of drug resistance in multi-drug resistant epithelial ovarian cancer cells induced by RNA interference in vitro].

作者信息

He Jing, Li Li, Tang Bu-jian, Zhang Wei, Li Dan-rong, Wang Qi

机构信息

Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.

出版信息

Zhonghua Fu Chan Ke Za Zhi. 2009 Sep;44(9):686-90.

PMID:20079182

Abstract

OBJECTIVE

To explore whether or not multi-drug resistance could be reversed by RNA interference the expression of Topo II alpha gene in epithelial ovarian cancer cell lines in vitro.

METHODS

(1) The best silent small interference RNA (siRNA) of Topo II alpha gene was designed and chose and cloned into psilencer4.1-CMV-neo vector. The psilencer4.1-CMV-neo-Topo II alpha was transfected into SKOV3/DDP cell, then Topo II alpha siRNA(+)SKOV3/DDP cells was incubated. (2) The Topo II alpha 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 cytometry and high performance liquid chromatography method before and after Topo II alpha RNA interference in cells.

RESULTS

(1) The Topo II alpha gene expression level in SKOV3/DDP cells could be inhibited after the plasmid DNA psilencer4.1-CMV-neo-Topo II alpha transfeced. The expression level of Topo II alpha mRNA in Topo II alpha siRNA(+)SKOV3/DDP and SKOV3/DDP cells were 0 and 0.92 +/- 0.08; the expression level of Topo II alpha protein in Topo II alpha siRNA(+)SKOV3/DDP and SKOV3/DDP cells were 0.51 +/- 0.04 and 1.95 +/- 0.09 (P < 0.01). (2) The multi-drug resistance index of Topo II alpha 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 G(0)/G(1) and G(2)/M phase cell in Topo II alpha siRNA(+)SKOV3/DDP cells were higher than that in SKOV3/DDP cells (P < 0.05). (4) The content of cisplatin in Topo II alpha 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 II alpha gene expression in cisplatin-resistant epithelial ovarian cancer cells.

摘要

目的

探讨RNA干扰上皮性卵巢癌细胞系中拓扑异构酶Ⅱα（TopoⅡα）基因的表达能否逆转多药耐药。

方法

（1）设计并筛选出TopoⅡα基因的最佳沉默小干扰RNA（siRNA），克隆至psilencer4.1-CMV-neo载体。将psilencer4.1-CMV-neo-TopoⅡα转染至SKOV3/DDP细胞，培养得到TopoⅡα siRNA(+)SKOV3/DDP细胞。（2）分别采用RT-PCR和蛋白质印迹法检测稳定转染细胞系中TopoⅡα mRNA和蛋白的表达。采用噻唑蓝比色法、流式细胞术和高效液相色谱法检测细胞TopoⅡα RNA干扰前后的耐药指数、细胞周期及顺铂细胞内含量。

结果

（1）转染质粒DNA psilencer4.1-CMV-neo-TopoⅡα后，SKOV3/DDP细胞中TopoⅡα基因表达水平受到抑制。TopoⅡα siRNA(+)SKOV3/DDP细胞和SKOV3/DDP细胞中TopoⅡα mRNA表达水平分别为0和0.92±0.08；TopoⅡα siRNA(+)SKOV3/DDP细胞和SKOV3/DDP细胞中TopoⅡα蛋白表达水平分别为0.51±0.04和1.95±0.09（P<0.01）。（2）TopoⅡα siRNA(+)SKOV3/DDP细胞的多药耐药指数显著低于SKOV3/DDP细胞（3.46比5.05，P<0.05）。（3）TopoⅡα siRNA(+)SKOV3/DDP细胞中G(0)/G(1)期和G(2)/M期细胞百分比高于SKOV3/DDP细胞（P<0.05）。（4）顺铂作用24小时后，TopoⅡα siRNA(+)SKOV3/DDP细胞内顺铂含量显著高于SKOV3/DDP细胞（分别为157.20 ng和63.99 ng，P<0.05）。