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Silencing MDR1 gene expression reverses resistance of drug-resistant leukemia HT9 cells to allicin
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Abstract:
Objective:To investigate the effects of short hairpin RNA (shRNA) expression plasmid on silencing of MDR1 gene in drug resistant H79 cells and thus reverse the drug resistance of human promyelocytic leukemia HT9 cells to allicin. Methods: shRNA fragment targeting MDR1 gene was designed and constructed to obtain pSilencer3.1-shMDR1 expression plasmid, which was then stably transfected into HT9 cells. The expression of MDR1 mRNA in HT9 cells was assayed by real-time PCR. The P-gp protein (encoded by the MDR1 gene) expression was assayed by Western blotting. The cell mortality rate of HT9 cells was determined by MTT method. After treated with allicin, the apoptosis of HT9 cells was observed by gel electrophorosis, cell ultrastructure changes were observed under a transmission electron microscope, and the cell cycle was detected by flow cytometry. Results: pSilencer3.1-shMDR1 expression plasmid targeting MDR1 was constructed successfully and then stably transfected into HT9 cells to form the HT9-shMDR1 cell line. The MDR1 mRNA (\[0.027±0.002\] vs \[0.110±0.005\]，P<0.01) and P-gp protein expressions (\[0.856±0.014\] vs \[1.454±0027\], P<005) were significantly decreased in HT9-shMDR1 cells. The IC50 of allicin to HT9-shMDR1 cells significantly decreased compared with that in untransfected HT9 cells (\[26.66±0.59\] vs \[52.75±0.64\] μg/ml，P<0.01). The relative drug resistance reversal rate of HT9-shMDR1 to allicin was (49.45±1.86)%. Compared with untransfected HT9 cells, DNA ladder was more obvious in HT9-shMDR1 cells treated with allicin, and the typical apoptotic body was found by electron microscopy. Allicin treatment did not affect the cell cycle distribution of untransfected HT9 cells and control plasmid transfected HT9 cells (HT9-neo cells). However, the ratios of allicin-treated HT9-shMDR1 cells were significantly decreased in the S phase (\[3140±2.13\]% vs \[53.80±1.87\]%, P<0.01\] and were significantly increased in the G2/M phase (\[35.62±206\]% vs \[9.37±2.09\]%, P<0.01\]. Conclusion: pSilencer3.1-MDR1 expression plasmid targeting MDR1 gene can inhibit the expression of MDR1 gene, therefore reversing drug resistance of HT9 cells to allicin.
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Project Supported:
Project supported by the Natural Science Foundation of Heilongjiang Province of China (No. C200624), the Science and Technology Research Project of Heilongjiang Education Bureau (No. 11511447, No. 12511611), and the Start-up Scientific Research Foundation for Young Teachers in Qiqihar University (No. 2011K-M38)
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