Original Articles
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Jan 28, 2009; 15(4): 431-440
Published online Jan 28, 2009. doi: 10.3748/wjg.15.431
Reversal of multi-drug resistance by pSUPER-shRNA-mdr1 in vivo and in vitro
Guang-Dong Pan, Jian-Qing Yang, Lv-Nan Yan, Guang-Ping Chu, Qiang Liu, Yi Xiao, Lin Yuan
Guang-Dong Pan, Jian-Qing Yang, Lv-Nan Yan, Guang-Ping Chu, Qiang Liu, Yi Xiao, Lin Yuan, Department of General Surgery, People’s Hospital of Liuzhou, Medical University of Guangxi, Liuzhou 545001, Guangxi Zhuang Autonomous Region, China
Author contributions: Pan GD and Yan LN contributed equally to this work; Pan GD, Yang JQ, Yan LN designed the research; Pan GD, Chu GP, Xiao Y, Yuan L performed the research; Yuan L analyzed the data; Pan GD, Yan LN, Yuan L wrote the paper.
Supported by National Natural Science Foundation of China, No. 31070925; Natural Science Foundation of Guangxi Zhuang Autonomous Region, No. 0728102
Correspondence to: Guang-Dong Pan, Department of General Surgery, People’s Hospital of Liuzhou, Medical University of Guangxi, Liuzhou 545001, Guangxi Zhuang Autonomous Region, China. pgdhx@126.com
Telephone: +86-772-2662657
Fax: +86-772-2823618
Received: May 10, 2008
Revised: June 29, 2008
Published online: January 28, 2009
Abstract

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 mdr1 into HepG2 cells. shRNA targeting sequence was designed to be homologous to the P-gp encoding MDR1 mRNA consensus sequence. pSUPER-shRNA/mdr1 was constructed using the enzyme-digested technique. HepG2/mdr1 cells were transfected with vectors of pSUPER-shRNA/mdr1 to measure their efficacy by real-time PCR for mdr1 mRNA, flow cytometry (FCM) for P-gp expression, and Rhodamine efflux, MTT method for HepG2/mdr1 function, respectively. In vivo, mice tumors were treated by injecting pSUPER-shRNA/mdr1 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/mdr1 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/mdr1 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/mdr1 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/mdr1 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.

Keywords: Hepatocellular carcinoma; Multi-drug resistance; RNA interference