RNA干扰技术逆转卵巢上皮性癌细胞多药耐药的研究

目的探讨应用RNA干扰(RNAi)技术逆转卵巢上皮性癌(卵巢癌)细胞多药耐药的可行性。方法将设计合成的针对多药耐药基因MDR1的特异性小分子干扰RNA(siRNA),用脂质体转染具有MDR1基因高表达的卵巢癌紫杉醇耐药细胞株OVCAR8/TR。用荧光定量RT-PCR技术和流式细胞仪分别测定转染前后细胞MDR1mRNA及糖蛋白P-gp表达的变化;三磷酸腺苷(ATP)生物荧光法检测转染前后细胞对顺铂、氟尿嘧啶、阿霉素和紫杉醇药物敏感性(以ATP抑制率判断)的变化情况。结果转染后24、48、72、96和120h,OVCAR8/TR细胞MDR1mRNA的抑制率分别为26·42%、84·00%、78·43%、45·85%和0;转染后48h MDR1mRNA的抑制率达到最高峰。转染后24、48、72、96和120h,OVCAR8/TR细胞P-gp的抑制率分别为16·71%、49·64%、85·23%、65·98%和9·44%;转染后72h P-gp的抑制率达到最高。转染MDR1siRNA后,能够明显提高OVCAR8/TR细胞对紫杉醇和阿霉素的敏感性,在紫杉醇的作用下,转染前后OVCAR8/TR细胞的ATP抑制率分别为(25·8±3·1)%和(78·0±9·8)%,转染前后比较,差异有统计学意义(P<0·05)。结论在OVCAR8/TR细胞中,针对MDR1合成的siRNA能够有效地抑制MDR1mRNA和P-gp的表达,并能恢复其对紫杉醇和阿霉素的敏感性。应用RNAi技术,能够逆转卵巢癌细胞对化疗药物的多药耐药。

Reversal of multi-drug resistance in ovarian cancer cell by RNA interference

Objective To investigate the effects of small interference RNA (siRNA) on the inhibition of MDR1 mRNA and P-gp expression of ovarian cancer cells with high expression of MDR1 gene, and reversal of drug resistance. Methods siRNA was synthesized and transfected into human ovarian cancer cell line OVCAR8/TR by liposome. The expression of MDR1 mRNA at different times after transfection was measured by real time RT-PCR and the P-gp expression was detected by flow cytometry. Adenosine triphosphate (ATP)-bioluminence assay was applied to check the drug sensitivity to four different chemotherapeutic agents before and after transfection. Results The suppression rates of MDR1 mRNA were 26.42%, 84.00%, 78.43%, 45.85% and 0 respectively at 24, 48, 72, 96 and 120 hours after transfection. The P-gp suppression rates were 16.71%, 49.64%, 85.23%, 65.98%, 9.44% respectively at 24, 48, 72, 96 and 120 hours after transfection. The maximal suppression rates of MDR1 mRNA and P-gp occurred at 48 and 72 hours after transfection respectively. ATP-bioluminence assay showed that OVCAR8/TR cells were sensitive to fluorouracil, resistant to cisplatin, doxorubicin (adriamycin) and paclitaxel (taxol). After siRNA treatment, OVCAR8/TR cells were sensitive to paclitaxel and doxorubicin, but the resistance to cisplatin could not be reversed. Conclusions RNA interference (RNAi) presents in human ovarian cancer cells. siRNA can effectively inhibit the expression of mRNA and P-gp of the multidrug resistance gene MDR1, and can reverse the drug resistance to chemotherapeutic agents which are transferred by P-gp.