Reversal of multi-drug resistance by pSUPER-shRNA-mdr1 in vivo and in vitro

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 chemotherapy with adriamycin 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.

Figure 1

shRNA/mdr1 expression plasmids digested by restriction enzyme Bag II and Hind III, and a fragment produced in 1.5% agarose gel electrophoresis. Lanes 1 and 7: positive clone size of 310 bp (insert element 64 bp).

Figure 2

Sequence of pSUPER-shRNA/mdr1 (inserted element 64 bp).

Figure 3

Real-time PCR showing significantly reduced mdr1 mRNA expression in cells treated with pSUPER-shRNA/mdr1 vector. 1: HepG2; 2: HepG2/mdr1; 3: HepG2/m-sh; 4: HepG2/mdr1 empty vector; M: Marker; Mdr1: 218 bp; GAPDH: 145 bp.

Figure 4

FCM showing P-gp expression of HepG2/mdr1 in test groups (A) and control groups (B).

Figure 5

Sensitivity of HepG2/mdr1 and HepG2/mdr1-siRNA to ADM. After transfected with pSUPER-shRNA/mdr1 vectors, the resistance of HepG2 cells to adiramycin and mytomycin decreased from 44.6-fold to 1.4-fold and from 138.1-fold to 1.14-fold.

Figure 6

DNR accumulation of HepG2/mdr1 (A, B) and HepG2 cells (C) pre- and post-transfection. Compared with control groups, accumulation of daunorubicin in cells treated with shRNA vectors increased significantly (79.32% vs 37.96%, P < 0.05). There was no difference in sensitive cells of HepG2 between test groups (79.32% vs 87.56%).

Figure 7

Changes in volume and growth ratio of tumor after treatment with ADM. More changes in tumor volume occurred in transfected groups than in control groups (1695.70 ± 152.54 mm³ vs 700.14 ± 25.61 mm³, P < 0.01).

Figure 8

FCM showing lower P-gp expression in HepG2 cells (A) and HepG2/mdr1-shRNA (B) in test groups than in control groups (65% vs 94.1%, P < 0.05). There was no significant difference between groups treated with in situ injection and intra-abdominal injection of pSUPER-mdr1 (65.1% vs 58.7%).

Figure 9

Immunohistochemistry showing green fluorescence in test groups (A) and control groups (B), P-gp expression in test groups (C) and control groups (D), P-gp expression in test groups transfected with in situs injection (E) and intra-abdominal injection of pSUPER-mdr1 (F) (× 200). The transfection ratio for test groups was higher than that for control groups (86.70% vs 35.20%, P < 0.05). The expression of P-gp in test groups was lower than that in control groups (15.75% vs 97.90%, P < 0.05).