反义MRP3寡核苷酸对卵巢癌拓普替康耐药性的逆转作用

MRP3 Antisense Oligodeoxynucleotide Can Reverse Resistance in Topotecan-resistant Ovarian Cancer Cells (SKOV3/TPT)

目的:探讨MRP3反义寡核苷酸对人卵巢癌拓普替康(TPT)耐药株SKOV3/TPT细胞的耐药逆转作用。方法:人工合成MRP3反义寡核苷酸(ASODN)及相应的正义寡核苷酸(SODN)片段,用脂质体(LF)构建成ASODN/LF、SODN/LF,分别转染SKOV3/TPT细胞,用MTT法、流式细胞仪(FCM)及半定量RT-PCR检测体外转染后细胞内TPT的荧光强度、耐药指数及MRP3mRNA表达的改变。结果:将MRP3反义及正义寡核苷酸片段分别转染进SKOV3/TPT细胞后,反义组细胞内TPT的荧光强度及耐药指数明显降低(P<0.05),细胞内MRP3mRNA的表达较未转染细胞下降了60.16%(P<0.05);正义组上述指标无明显变化。结论:转染MRP3ASODN/LF可部分逆转MRP3介导的人卵巢癌细胞对TPT耐药。

Objective： To investigate the reversal of topotecan resistance by MRP3 antisense oligonucleotide （ASODN） on topotecan-resistant SKOV3/TPT cells. Methods： A 22-base antisensephosphorothioate oligonucleotide for MRP3 was artificially synthesized that included the translation initiation site. The sense oligonucleotide （SODN） corresponding to the ASODN was also synthesized as a control. Lipofect-2000 （LF） was used for the transfer of either ASODN or SODN into SKOV3/TPT cells. The resistance index of topotecan, intracellular fluorescence intensity of TPT and changes of MRP3 mRNA expression in transfected SKOV3/TPT cells were detected by methyl thiazolyl tetrazolium （MTT） assay, flow cytometry （FCM）, and semiquantitative reverse transcription-polymerase chain reaction （RT- PCR）, respectively. Results： The transfer of MRP3- ASODN/LF into SKOV3/TPT cells resulted in： （1） a decreased resistance index to topotecan from 10.67 to 3.165. （2） an obviously increased intracellular TPT fluorescence intensity from 4.55±0.75 to 9.05±13 （P〈0.05）; （3） Reduction of MRP3 mRNA level （60.16%）（P〈0.05） in SKOV3/TPT cells, as compared with non-transfected cells. No significant change could be detected after transfecting SODN or LF alone. Conclusion： ASODN transfection may partly reverse MRP3-mediated drug- resistance of ovarian cancer cells to TPT.