Reversal Of Multidrug Resistance Of Hepatocellular Carcinoma With Combination Silent The Mdr-1and Mrp-1by Sirna Technique In Vitro

Objective:To explore the different effect between Combination Silent the mdr-l,mrp-1and separateness Silent in reversal of multidrug resistance of hepatocellular carcinoma by siRNA Technique.Methods:We designed sequence of siRNA produced based on the experimental results of screening high efficiency siRNA.64bp oligonucleotides of pSUPER the target sequence of siRNA/mdr-1and siRNA/mrp-1were synthesized and annealed to form duplex strand,then were cloned into pSUPER to construct siRNA/mdr-1vector and siRNA/MRP-1vector. Competent E.coil was transfected by vector of pSUPER-siRNA/mdr-1and SUPER-siRNA/mrp-1to screen the positive clones for sequencing and extracting plasmid. the plasmids extracted were used to transfect HepG2/MDR cells with control groups by negative vectors.The expression of mRNA was measured by real-time PCR, and Flow cytometry was used to study the effect on cell cycle.Results:The pSUPER-siRNA/mdr-1and pSUPER-siRNA/mrp-1were established successfully and was sequenced to test its accuracy.(1)The expression of MRP-mRNA in HepG2/MM-si was the same as that in HepG2/mrp-1-siRNA (P>0.05). The expression of MDR-mRNA in HepG2/MM-si was the same as that in the HepG2/mdr-l-siRNA (P>0.05).(2)The activation of Caspase3in HepG2/MM-siRNA was higher than that in HepG2/mrp-1-siRNA (14623.7±338.9vsl3215.7±90.6, p<0.05); but Caspase-3in HepG2/MM-siRNA was the same as that in HepG2/mdr-1-siRNA.(3)The cytoactive after5-FU administration the in HepG2/MM-siRNA was lower than that in HepG2/mdr-l-siRNA and HepG2/mrp-1-siRNA.(4)Compared to positive compared experiments, the relative resistant folds of HepG2/mdr-1-si and HepG2/mrp-1-si were1.39folds,1.58folds respectively.(5) The results of analysis cell cycle and apoptosis indicated that the HepG2/MM-siRNA cells were less than the HepG2/mdr-l-si and HepG2/mrp-1-si.Conclusions:(1)The vectors of pSUPER-HepG2/mdr-l-si and pSUPER-HepG2/mrp-l-si can be constructed by technique of enzymatic incison. Different vector of siRNA transfection into the HepG2/MDR at the same time will not interference each other.(2) In view of existing situation that several genes lead to multidrug resistance of hepatocellular carcinoma promote a diversified siRNA sequences to silent various drug-resistant genes is inevitable trend of the development of Therapy for Liver Cancer.