Study On Reversal Of Multidrug Resistance On B-MD-C1(ADR +/+) By RNA Interference On The Suppression Of CtBP1 Gene

Objective: Design and construct the eukaryotic expression vector of human CtBP1 gene short hairpin RNA (shRNA) then to transfect human endometrial carcinoma cells B-MD-C1(ADR^+/+). To investigate the effect of CtBP1 gene silencing mediated by RNA interference on the expression of MDR1 and P-gp in human endometrial carcinoma cells B-MD-C1(ADR^+/+) and the sensibility of B-MD-C1(ADR^+/+) cells to ADM, and to explore the role of CtBP1 gene on reverse the cancer multidrug resisrence.Methods: 1 MDR was investigated by using cell culture technique and MTT colorimetric method and to determine the experiment conditions.2 Expression of MDR1 in B-MD-C1(ADR^+/+) cells and B-MD-C1(wt) cells was detected by RT-PCR.3 Expression of P-gp in B-MD-C1(ADR^+/+) cells and B-MD-C1(wt) cells was detected by Western blot.4 To design and construct the eukaryotic expression vector of human CtBP1 gene short hairpin RNA (shRNA) then to transfect into B-MD-C1(ADR^+/+) cells with Lipofectamin TM 2000.5 Expression of MDR1 in B-MD-C1(ADR^+/+) cells and the cells after transfected at 48 hour was detected by RT-PCR.6 Expression of P-gp in B-MD-C1(ADR^+/+) cells and the cells after transfected at 72 hour was detected by Western blot.7 Sensibility of B-MD-C1(ADR^+/+) cells and the cells after transfected at 0,24,48,72 hour to ADM was evaluated by means of MTT colorimetric method.Results: 1 The IC50 value of B-MD-C1(ADR^+/+) cells to ADM were 21.57±0.77μg/ml. While the IC50 value of B-MD-C1(wt) cells to ADM were 2.32±0.34μg/ml. The MDR of B-MD-C1(ADR^+/+) cells was higher 9.3 times than B-MD-C1(wt) cells (p<0.01).2 Expression of MDR1 in B-MD-C1(ADR^+/+) cells was stronger than in B-MD-C1(wt) cells (p<0.01).3 Expression of P-gp in B-MD-C1(ADR^+/+) cells was stronger than in B-MD-C1(wt) cells (p<0.01).4 Two eukaryotic expression vector of human CtBP1 gene short hairpin RNA (shRNA) were constructed and could effectively inhibit the expression of CtBP1 gene by RNAi (inhibition rate were 47.18% and 41.75%).5 Silencing of CtBP1 expression by the targeting siRNA remarkably decreased the amount of MDR1 mRNA in B-MD-C1(ADR^+/+) cells after transfection with p eukaryotic expression vector for 48 hour.6 Expression of the MDR1 gene product, P-gp, was also reduced with the knockdown of CtBP1 after transfection with eukaryotic expression vector for 72 hour.7 The proliferation was significantly inhibited after ransfection with eukaryotic expression vector for 24,48,72 hour, especially for 48 and 72 hour (p<0.01).Conclusion: 1 Human carcinoma uterus cell line B-MD-C1(ADR^+/+) showed resistance to ADM and one of mechanisms is over-expression of P-gp.2 The eukaryotic expression vectors of CtBP1 shRNA could effectively inhibit the expression of CtBP1 gene.3 The eukaryotic expression vectors of CtBP1 shRNA could down-regulate the expression of MDR1 mRNA and P-gp.4 The eukaryotic expression vectors of CtBP1 shRNA could increase sensitivity of B-MD-C1(ADR^+/+) to AMD.