AIM： To explore the possibility of reversing multi-drug resistance （MDR） to HepG2/mdr1 in vitro and in vivo with RNA interference （RNAi）. METHODS： HepG2/mdr1 was obtained by cloning the whole gene mdrl into HepG...AIM： To explore the possibility of reversing multi-drug resistance （MDR） to HepG2/mdr1 in vitro and in vivo with RNA interference （RNAi）. METHODS： HepG2/mdr1 was obtained by cloning the whole gene mdrl into HepG2 cells, shRNA targeting sequence was designed to be homologous to the P-gp encoding MDR1 mRNA consensus sequence, pSUPER- shRNA/mdrl was constructed using the enzyme- digested technique. HepG2/mdrl cells were transfected with vectors of pSUPER-shRNA/mdrl to measure their efficacy by real-time PCR for mdrl mRNA, flow cytometry （FCM） for P-gp expression, and Rhodamine efflux, MTT method for HepG2/mdrl function, respectively. In vivo, mice tumors were treated by injecting pSUPER-shRNA/mdrl in situ and into intra- abdominal cavity. Tumors were collected to create cell suspension and cryosections after chemothearpy with adiramycin and mytomycin. The cell suspension was incubated in RPMI-1640 supplemented with G418 to screen stable cells for appreciating the reversal of MDR. Cryosections were treated with immunohistochemistry technique to show the effectiveness of transfection and the expression of P-gp.RESULTS： pSUPER-shRNA/mdrl was successfully constructed, which was confirmed by sequencing. The MDR phenotype of HepG2/mdr1 was decreased significantly in vitro transfection. HepG2/mdr1 showing its MDR was reversed notably in P-gp expression （11.0% vs 98.2%, P 〈 0.01）. Real-time PCR showed that mRNA/mdrl was lower in test groups than in control groups （18.73± 1.33 vs 68.03±2.21, P 〈 0.001）. Compared with HepG2, the sensitivity of HepG2/ mdrl and HepG2/mdr1-dsRNA cells to ADM was decreased by 1.64 times and 15.6 times, respectively. The accumulation of DNR in positive groups was decreased evidently. In vivo, the p-gp expression in positive groups was significantly lower than that in control groups （65.1% vs 94.1%, P 〈 0.05）. The tumor suppressing rate in test groups was 57.8%. After chemotherapy, the growth rate in test groups was lower than that in control groups （700.14 ± 35.61 vs 1659.70 ± 152.54, P 〈 0.05）. Similar results were also observed under fluorescence microscope, and confirmed by Image-Pro Plus 4.5 analysis. CONCLUSION： pSUPER-shRNA/mdrl vector system allows simple, stable and durable nonviral knockdown of P-gp by RNAi in malignant cells and animals to restore their sensitivity to adriamycin.展开更多
基金Supported by National Natural Science Foundation of China, No.31070925Natural Science Foundation of Guangxi Zhuang Autonomous Region,No.0728102
文摘AIM： To explore the possibility of reversing multi-drug resistance （MDR） to HepG2/mdr1 in vitro and in vivo with RNA interference （RNAi）. METHODS： HepG2/mdr1 was obtained by cloning the whole gene mdrl into HepG2 cells, shRNA targeting sequence was designed to be homologous to the P-gp encoding MDR1 mRNA consensus sequence, pSUPER- shRNA/mdrl was constructed using the enzyme- digested technique. HepG2/mdrl cells were transfected with vectors of pSUPER-shRNA/mdrl to measure their efficacy by real-time PCR for mdrl mRNA, flow cytometry （FCM） for P-gp expression, and Rhodamine efflux, MTT method for HepG2/mdrl function, respectively. In vivo, mice tumors were treated by injecting pSUPER-shRNA/mdrl in situ and into intra- abdominal cavity. Tumors were collected to create cell suspension and cryosections after chemothearpy with adiramycin and mytomycin. The cell suspension was incubated in RPMI-1640 supplemented with G418 to screen stable cells for appreciating the reversal of MDR. Cryosections were treated with immunohistochemistry technique to show the effectiveness of transfection and the expression of P-gp.RESULTS： pSUPER-shRNA/mdrl was successfully constructed, which was confirmed by sequencing. The MDR phenotype of HepG2/mdr1 was decreased significantly in vitro transfection. HepG2/mdr1 showing its MDR was reversed notably in P-gp expression （11.0% vs 98.2%, P 〈 0.01）. Real-time PCR showed that mRNA/mdrl was lower in test groups than in control groups （18.73± 1.33 vs 68.03±2.21, P 〈 0.001）. Compared with HepG2, the sensitivity of HepG2/ mdrl and HepG2/mdr1-dsRNA cells to ADM was decreased by 1.64 times and 15.6 times, respectively. The accumulation of DNR in positive groups was decreased evidently. In vivo, the p-gp expression in positive groups was significantly lower than that in control groups （65.1% vs 94.1%, P 〈 0.05）. The tumor suppressing rate in test groups was 57.8%. After chemotherapy, the growth rate in test groups was lower than that in control groups （700.14 ± 35.61 vs 1659.70 ± 152.54, P 〈 0.05）. Similar results were also observed under fluorescence microscope, and confirmed by Image-Pro Plus 4.5 analysis. CONCLUSION： pSUPER-shRNA/mdrl vector system allows simple, stable and durable nonviral knockdown of P-gp by RNAi in malignant cells and animals to restore their sensitivity to adriamycin.
