Reversal Of Multidrug Resistance By ShRNA In Human Bladder Cells BIU-87/ADM

【Objective】: To construct a short hairpin RNA (shRNA) eukaryotic expression vector specific to MDR1 gene by pSIREN-RetroQ-ZsGreeen vector, and Research whether the shRNA can reverse the expression of MDR1 gene in human bladder line BIU-87/ADM.【Methods】: According to BarnH I + Sense + Loop + Antisense + Termination signal + EcorI structure , Two pairs of oligos for shRNA expression which targeted MDR1 gene were chemically synthesized,and the eukaryotic expression vectors of shRNA aiming at MDR1 mRNA Target sequences were constructed by gene recombination. then transfected into the multidrug resistance bladder cells BIU-87/ADM with liposome- induced Gene transfection . Expression of MDR1 mRNA was assessed by RT-PCR, p-gp expression was determined by immunocytochemistry, and the sensitivity of cell lines to doxorubicin (ADM) was detected by MTT test.Finally, The results were analysised by Using SPSS,groups all using two-sample t-test。【Results】: Recombinant plasmid pSIREN - RetroQ - ZsGreeen - A and pSIREN - RetroQ - ZsGreeen– B were identified by PCR and confirmed by sequencing analysis,The results indicated that two pairs of oligos had been inserted into the expected site.。Furthermore, the inserted sequence were exactly correct without any base mutation。Two recombinant plasmid were transfected into the multidrug resistance bladder cells BIU-87/ADM. After 24 hours,In BIU-87/ADM transfected with pSIREN-RetroQ-ZsGreeen-A and pSIREN- RetroQ-ZsGreeen–B,RT-PCR showed that MDR1 mRNA expression was significantly inhibited, expression of MDR1 mRNA was reduced by 72.57% and 78.88% respectively(p﹤0.05); After 48 hours immunocytochemistry showed that the P-gp positive expression rate was 48.53±2.65% and 43.58±2.52 %respectively(p﹤0.05), There was significantly differece between untransfe -cted group and tansfected group. Then, MTT assay indicated that the transfected BIU-87/ADM cells showed an enhanced sensifivity to ADM,the relative efficiency of BIU-87/ADM to ADM was 52.02% and 54.87% respectively(p﹤0.05)。Furthermore, there was not differece between pSIREN-RetroQ-ZsGreeen-A and pSIREN-RetroQ-ZsGreeen-B in the suppression of MDR1 expression。【Conclusion】: The shRNA expression vector targeting MDRl gene showed dramatic inhibition on RNA transcription and protein expression.It can restore the sensitivity of BIU-87/ADM line to conventional ehemotherapeutie agents, therefore, may provide a kind of new means in the treatment of multidrug resistance of bladder cancer.