Stable Reversal Of Multidrug Resisitance In A549/DDP Cells By RNAi Targeting MRP1 Gene

AIM:To reverse the multidrug resistance by RNA interference (RNAi)-mediated MRP1 suppression in A549/DDP cells.METHODS: Four templaes sequence for siRNA against cDNA sequence of mrp1gene were designed and two MRP1 shRNA expression plasmid pSilencer 2.1-U6 neo-mrp1 were constr ucted. . In dividual bacterialclonies PCR and sequencing were performed to verify the recombine ation vector to confirm the better one which have no mutation.We transfected the A549/DDP cell by transformed plasmid.Two positive clones were selected by G418. Then selected the stable tran sfected cells to culture.The MRP function was determined by rho123 retention .The expression of MRP1 protein was detected by FCM and immunofluorescence.MRP1 mRNA was assayed by rea l -time fluorescent quantitative PCR.The efficiency of A549/DDP was determined by the MTT method. Then selected the stable trans fected cells,detected inhibition efficiency by methords of Flow cytometry (the detection of apoptotis rate and cell cycle,Acridine-Orange Staining).RESULTS:1.pSicencer-2.1-1 and pSicencer-2.1-2 were constructe using pSicenc er-2.1-U6 neo.2 We transfected the A549/DDP cell by transformed plasmid and two positive clones were suggested.3.The results of Flow cytometry and real-time fluorescent quantitative PCR showed that the MRP1 mRNA was inhibited in different degree. The MRP1 mRNA was significantly descreased to52.11±0.00%,44.97±0.81% in A549/DDP/sh-MRP1-p2.1-1-1and A549/DDP/sh-MRP1-p2.1-2-1 cells and the expression rate of MRP1 protein were 59.83±1.44%,36.19±0.74%. the expression rate of MRP1 protein were decreased seriously by immufluorescence .The first shRNA was superior to the second one , apoptotis rate was increated and cell cycle was changed.4. Rhodamine efflux shwmed that efflux function of MRP1 in transfected cells decreased in different degree.5. chemosensitivity of transfected cells to DDP increased in different degree, the relative rate reversal in A549/DDP/sh -MRP1-p2.1-1-1 and A549/DDP/sh-MRP1-p2.1-2-1 cells were 79.35±1.86%,59.74±0.64% ,chemosensitivity of transfected cells to 5-FU increased in different degree, the relative rate reversal in A549/DDP/sh-MRP1-p2.1-1-1 and A549/ DDP/sh-MRP1-p2.1-2-1 cells were 78.55±0.59%, 57.93±1.80%.6.The A549/ DDP/sh-MRP1-p2.1-1-1 and A549/DDP/sh-MRP1-p2.1-2-1 cells was a apoptoted seriously using fluorescence microscope and agrose gel.CONCLUSION: The two shRNA inhibited the expression of MRP1 gene in different degree,and reversed the drug resistance of A549/DDP cells in different degree, the first shRNA was superior to the second one , the reversal effect of the A549/DDP monoclone cells treated by siRNA were different.