Study On The Correlation Of GCS And MDR1 In Inducing Multidrug Resistance In Human K562/A02 Cell Line

Objective To investigate the correlation between Glucosylceramide synthase(GCS)and multidrug resistance 1(MDR1)in inducing multidrug resistance in human multidrug-resistant K562/A02 cell line, and search a novel strategy for reversing multidrug resistance of leukemia cells.Methods (1) By RT-PCR, the expression levels of GCS and MDR1 mRNA in K562 and K562/A02 cells were assayed. (2) Two kinds of siRNAs targeted at GCS gene and MDR1 gene were transfected into K562/A02 cells with liposome respectively. Forty-eight hours later, the differential expressions of GCS and MDR1 mRNA as well as correlation between them were detected by RT-PCR and Real-time PCR. (3) To further investigate the correlation of mRNA of GCS and MDR1, twenty-four hours after siRNA targeted at GCS gene was transfected into K562/A02 cells with liposome, the mRNA of GCS and MDR1 were detected by Real-time PCR. (4) Seventy-two hours after the siRNAs targeted at GCS and MDR1 were transfected into K562/A02 cells respectively, total protein were extracted and the expression levels of P-glycoprotein were analyzed by Western-blot .Results (1) The expression levels of GCS and MDR1 mRNA were dramatically lower in drug-sensitive K562 cells compared with K562/A02 cells. (2) The expression level of GCS mRNA was inhibited by 73%(59~82% )and MDR1 mRNA expression was down regulated by 67% (38~82% ) in K562/A02 cells at 48 hours after being transfected GCSsiRNA. At same time, the expression level ofMDR1 mRNA was inhibited by 81%(63~91% )and GCS mRNA expression had no apparent change in K562/A02 cells which were treated with MDR1siRNA. (3) Twenty-four hours after GCSsiRNA was transfected into K562/A02 cells, the expression level of GCS mRNA was inhibited by 69%(58%～78%) and MDR1mRNA expression had no apparent change. Compared with 24 hours after GCSsiRNA was transfected into K562/A02 cells, there was no obviously various expression of GCS mRNA whereas MDR1mRNA was down regulated by 65%(54%～74%) at 48 hours. (4) Seventy-two hours after the siRNAs targeted at GCS and MDR1 were transfected into K562/A02 cells respectively, the expression levels of P-glycoprotein decreased by 1.44(1.35～1.53)and 3.54(3.44～3.65)times.Conclusion There was positive correlation between GCS and MDR1 mRNA in K562/A02 cells . The expression of MDR1 mRNA and P-glycoprotein was down regulated by silencing GCS mRNA. Moreover, the down regulation was insecondary and dependent on time-effect. Thereby we conclude that there is a high correlation GCS and MDR1 in inducing multidrug resistance in human K562/A02 cell line.