Construction of expressing vector of pSUPER-shRNA/mrp1 and its expression in vitro / 中华肝胆外科杂志

ABSTRACT

Objective To construct the expressing vector of shRNA/mrp1 and study its expression in vitro. Methods 64bp oligonucleotides of pSUPER and the targeted senquence of siRNA/mrp1 were synthesized and annealed to form duplex strand, then were cloned into pSUPER to construct pSUPER-shRNA/mrp1 vector. Competenced Ecoli was transfected by vector of pSUPER-shRNA/ mrp1 to screen the positive clones for sequencing and extracting plasmid. The plasmids extracted were used to transfected HepG2/mrp1 cells with a control groups by negative vectors. The expression of mrp1 mRNA and MRP1 was measured by real-time PCR and resistance of HepG2/mrp1 by flowcytometry. Results pSUPER-shRNA/mrp1 was established successfully and was sequenced to test its accuracy. Expression of mrp1 mRNA in HepG2/mrp1-si was lower than that in HepG2/mrp1 (1-fold vs 179.76-fold, P<0.001). Compared to HepG2/mrp1, the expression of MRP1 in HepG2/mrp1-si was lower (11.2% vs 97.6%, P<0. 05). The sensitivity of HepG2/mrp1-si to adiramycin was higher than that of HepG2/mrp1(45.0-fold vs 1.2-fold, P<0.01). Meanwhile, the accumulation of DNR in HepG2/mrp1-si increased significantly as compared with the control (78.58 % vs 38.44%,P<0.05).Conclusion Vector of pSUPER-shRNA/mrp1 can be constructed by the technique of enzymatic incision. The multidrug resistance of HepG2/mrp1 can be reversed by RNA interference.