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Effect of GCS on Expression of MDR1mRNA and P-gp Function in K562/AO2 Cell Line

Author: ZhangYangYang
Tutor: XiePing；XieKeMing
School: Suzhou University
Course: Pathology and Pathophysiology
Keywords: glucosylceramide synthase multidrug resistance gene 1 P-glycoprotein, rhodamine123 small interfering RNA leukemia real-time PCR mean fluorescence intensity
CLC: R733.7
Type: Master's thesis
Year: 2011
Downloads: 29
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Abstract


Objective To explore the effect of glucosylceramide synthase (GCS) on MDR1 and P-gp drug-efflux in K562/AO2 leukemia drug-resistance cell line, study the molecular mechanism of GCS inducing drug-resistance in K562/AO2 leukemia cell line.Methods (1) The IC50 (the concentration causing 50℅ inhibition of cell growth) of ADR to K562, K562/AO2 cells and RNA interfering group which had been treated by siRNA were assayed by MTT method. (2) Two kinds of siRNAs targeted at GCS gene and MDR1 gene were transfected into K562/A02 cells respectively. Forty-eight hours later, the expression level of GCS and MDR1 mRNA were detected by Real-tine PCR. (3) After being transfected GCSsiRNA into K562/AO2 cells for 9 and 36 h respectively, the expression level of GCS and MDR1 mRNA were detected by Real-tine PCR. (4) Two kinds of siRNAs targeted at GCS gene and MDR1 gene were transfected into K562/A02 cells respectively. Forty-eight hours later, the dynamic mean fluorescence intensity (MFI) which represents intracellular rhodamine 123 retention (reflect P-gp function) was analyzed by Flow Cytometric.Results (1) MTT assay showed that IC50 of doxorubicin to K562/AO2 cells (138.25±3.75ug/ml) was significantly higher than that of K562 drug-sensitive cells (2.125±0.125ug/ml), and the IC50 was evidently lower in K562/AO2 cells, whether it was transfected with GCSsiRNA or MDR1siRNA. (2) Transfected siRNA into cells for 48 h, the results showed that the mRNA inhibition rate of GCSsiRNA to GCS and MDR1siRNA to MDR1 was 68% (63~76%) and 75 % (74~81%), respectively. (3) the inhibition rates of GCS mRNA in the cells transfected with GCSsiRNA for 9 h and 36 h were 64%(60～66%) and 71%(69～75%)respectively. Interestingly, the expression of MDR1 mRNA was also inhibited to 51%(46～57%)after being transfected with GCSsiRNA at 36 h, but there was no significant difference in MDR1 expression at 9 h post-transfection in cells treated with GCSsiRNA. (4) Rh123 retention of K562 cells was very higher than K562/AO2 cells. Whatever GCS or MDR1 RNA interfering group, rhodamine123 retention significantly increased compared with the negative control group after RNAi for 48 h.Conclusion Our works confirmed inhibiting GCS gene could down-regulated the expression of MDR1 mRNA and P-gp efflux function, this suggested that GCS may contributed to drug- resistance of human leukemia cell by cooperating with P-gp.

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