Knockdown Of HOXA10 Combined Tetramethylpyrazine Reverses Multidrug Resistance Of K562/ADM Cells And Its Molecular Mechanism

Objective: To investigate the effect of silencing HOXA10 gene by RNA interference combined Tetramethylpyrazine on reversing multi-drug resistance and its molecular mechanism in K562/ADM cells,find new therapeutic strategy for reversing MDR in leukemia clinical treatment.Methods:1. The pGPHI/GFP/Neo/HOXA10 shRNA eukaryotic expression vector based on the best HOXA10 siRNA fragments screened out in previous experiments was constructed after enzyme digestion and sequence identification.The eukaryotic expression vector was stably transfected with K562/ADM cells.Then the effect of silencing HOXA10 gene by eukaryotic expression vector was determined by Real-time RT-PCR and Western blotting analysis. The reversal effect was measured by MTT assay after silencing HOXA10 gene. Intracellular Adr accumulation was determined by flow cytometry and expressions of P-gp and MRP1 were determined by Western blotting assay.2. The reversal effect of TMP was measured by MTT assay. Intracellular Adr accumulation was determined by flow cytometry and expressions of P-gp and MRP1 were determined by Western blotting assay.3. The reversal effect was measured by MTT assay after silencing HOXA10 gene combined TMP.Intracellular Adr accumulation was determined by flow cytometry and expressions of P-gp and MRP1 were determined by Western blotting assay.Results:1.The expression vector targeting HOXA10 gene(pGPHI/GFP/Neo/HOXA10 shRNA) was created successfully by enzyme digestion.The pGPHI/GFP/Neo/HOXA10 shRNA eukaryotic expression vector was stably expressed in K562/ADM cells.The expression of HOXA10 mRNA and HOXA10 protein were( 38.6±5.95)% and( 21.45± 1.143)% respectively after transfected with pGPHI/GFP/Neo/HOXA10 shRNA eukaryotic expression vector.The reversal fold of HOXA10 interference group was 2.05.Adriamycine intracellular mean fluorescence was(315.33±3.511).P-gp and MRP1 relative expression of K562/ADM cells transfected with pGPHI/GFP/Neo/HOXA10 shRNA eukaryotic expression vector were (35.74±6.34)%and(39.43±3.144)%respectively(P<0.05).2.The cell growth inhibition of 1μg/ml and 2μg/ml TMP was(2.1434±0.17263)% and (8.6034±0.33859) respectively.The reversal fold of 1μg/ml,2μg/ml TMP were 1.15 and 1.27 respectively.The intracellular accumulation of Adr of 1 μg/ml was (237±2.57) ,while 2μg/ml TMP was(273.33±4.509) .Both 1 μg/ml and 2pμg/mlTMP inhibited the expression of P-gp and MRP1 in a dose-dependent manner. ( P <0.05)3.The reversal fold and intracellular accumulation of Adr of Tetramethylpyrazine+HOXA10 RNA interference group were 5.08、(454.33±6.11),which were higher than HOXA10 RNA interference group and Tetramethylpyrazine group.Silencing HOXA10 gene combined Tetramethylpyrazine decreased expressions of P-gp and MRP1 to(19.85±8.347) % and (20.44±3.311) % (P<0.05).Conclusions:1. pGPHI/GFP/Neo/HOXA10 shRNA eukaryotic expression vector inhibited HOXA10 mRNA and HOXA10 protein expression effectively.Both silencing HOXA10 gene and TMP reversed the MDR of K562/ADM cells.TMP reversed the MDR in a dose-dependent manner.3.Silencing HOXA10 gene combined TMP could significantly inhibited expressions of P-gp and MRP1, increased Adr intracellular accumulation of K562/ADM cells,thus reverse K562/ADM cells MDR.Silencing HOXA10 gene combined TMP reversed MDR of K562/ADM cells significantly and they can act with each other.