Mdr1-mediated ShRNA Construction Of Expression Plasmid And Study On It Reversing The Multidrug Resistance Of Renal Carcinoma Cell ACHN

Objective:To construct two expressing plasmids of mdr1-mediated shRNA (short hairpin RNA,shRNA by pSIREN-RetroQ-ZsGreee(pRZ) vector;against the MDR1 gene over-expression mechanisms,research whether two different interference fragments can reverse the multi-drug resistance in human renal carcinoma ACHN。METHODS:1)According to BarnH I + Sense + Loop + Antisense + signal + EcorI structure ,Two pairs of oligos for shRNA expression which targeted MDR1 gene were chemically synthesized, then annealed and phosphorylated. Finally, annealed and phosphorylative oligos were inserted into pRZ vector to construct RNAi plasmid。2)Renal carcinoma cell ACHN were incubated in vitro,RNAi plasmids were transfected into ACHN cells by LyoVecTM。3)The sensitivity of cell lines to doxorubicin (ADM) was detected by MTT test;the P-gp expression was detected by Immunohistochemistry;The mRNA of mdrl gene was identified by Semiquantitative RT-PCR.4)The data was analysised by Using SPSS16.0,The data was showed by x±S,groups all using two-sample t-test,it has Significant difference when p<0.05。RESULTS:Recombinant plasmid pRZ-A,pRZ-B (pSIREN - RetroQ - ZsGreeen - A and pSIREN - RetroQ - ZsGreeen - B) was 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 was exactly correct。the ACHN green fluorescent protein expression was observed and counted under inverted microscope after transfected pRZ-A and pRZ-B 24h,48h,96h。In which transfection 48h had the strongest expression of green fluorescent protein,the transfection efficiency, respectively is 75.35±1.15%,74.83±1.37%;MTT assay showed that drug sensitivity was increased significandy after pRZ-A and pRZ-B vectors was transfected into ACHN cells,ACHN blank control group IC50 is about 2.5ug/ml, ACHN blank control group IC50 is about 2.5ug/ml, transfected pRZ-A, pRZ-B ACHN IC50 decreased to 0.65ug/ml,0.7ug/ml,respectively,compared with the blank control group decreased by 83.44%,83.42%;The P-gp positive expression rate has significantly differece (t = 15.621 P <0.05) between transfected pRZ-A 96h cell and the blank control group,decreased by 44.69%。The P-gp positive expression rate has significantly differece between transfected pRZ-B 48h cell(t=2.415 P<0.05) and the blank control group,decreased by 7.32%。The P-gp positive expression rate has significantly differece (t = 17.995 P <0.05) between transfected pRZ-B 96h cell and the blank control group, decreased by 53.62%。Compared with untransfected group,Semi-quantitative RT-PCR show that the transfected pRZ-A or pRZ-B cell MDRl mRNA was significanfly reduced(P<0.05),decreased by 82.5% and 77.5%;There was no differece between pRZ-A and pRZ-B in the suppression of MDR1 expression。CONCLUSION : Successfully construction of expressing plasmid of mdr1-mediated shRNA。pRZ-A and pRZ-B vector can effectively reverse the renal carcinoma cell ACHN multidrug resistance,improve the sensitivity to ADM, inhibit P-gp-mediated multidrug resistance。...