[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].

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.