Molecular Mechanisms Investigation Into Adriamycin Resistance Mediated By CD44 Gene In Leukemia K562 Cells

Objective:To investigate the possible molecular mechanism into adriamycin resistance mediated by CD44 gene in leukemia K562 cells;to elucidate the correlation between CD44 gene and adriamycin resistance of K562 cells;to explore the target gene that reversed the drug resistance in leukemia.Methods:1.After the escherichia coli containing CD44 plasmid was amplificated,that the expression of CD44 plasmid was successful or not was detected by Agarose gel electrophoresis assay;2.The extracted CD44 carrier plasmids were transferred into K562 cells to prepare the K562/CD44 cells which existed high expression of CD44 by electrical transfection assay;3.The green fluorescent protein of CD44 in K562 cells and K562/CD44 cells were detected by an inverted fluorescence microscope,the protein expression levels of CD44 were detected by Western blot assay,and the mRNA expression levels of CD44 were tested by Real-time fluorescence quantitative PCR assay;4.The mRNA expression levels in K562 cells and the K562/CD44 cells of drug resistance gene P-gp were tested by Real-time fluorescence quantitative PCR assay;5.After K562 cells and K562/CD44 cells were treated with 1.0mg/L Adriamycin for 48h,the proliferation rate of K562 and K562/CD44 cells were detected at the same concentration and action time by WST-1 cell proliferation assay;6.After treatment with 1.0mg/L Adriamycin for 24h,the cell apoptosis rate of K562 cells and K562/CD44 cells were detected respectively by Annexin V-FITC/PI double staining flow cytometry;7.The migration-related protein MMP-2 and MMP-9 as well as apoptosis-related protein Bax and Bcl-2 expression levers in K562 cells and K562/CD44 cells were detected by Western blot assay.Results:1.Agarose gel electrophoresis assay results showed that the plasmids which were extracted after the cultivation of escherichia coli showed a clear and bright band;2.The inverted fluorescence microscopy results manifested that the K562/CD44 cells which were prepared by transferring extracted CD44 plasmids into K562 cells had high expression of green fluorescent protein;3.Real-time fluorescence quantitative PCR assay results showed that CD44 and drug resistance gene P-gp mRNA expression levels in K562/CD44 cells were significantly higher than K562 cells(P<0.01);4.WST-1 cell proliferation assay results showed that after K562 cells and K562/CD44 cells treatment with 1.0mg/L Adriamycin for 48h,the proliferation rate of K562/CD44 cells was markedly increased compared with K562 cells(P<0.01);5.The results of flow cytometry manifested that the apoptosis rate of K562/CD44 cells treatment with 1.0 mg/L Adriamycin for 48 h was significantly decreased compared with K562 cells(P<0.01);6.Western blot results showed that the protein expression levels of CD44,MMP-2,MMP-9,Bcl-2/Bax in K562/CD44 cells were increased significantly(P<0.05 or P<0.01),and Bax were decreased significantly(P<0.01).Conclusions:1.The overexpression of CD44 resisted the inhibitory effect of adriamycin on K562 cells proliferation;2.The overexpression of CD44 may be involved in the drug resistance process of K562 cells by regulating drug resistance gene P-gp,migration-related protein MMP-2 and MMP-9 and apoptosis-related protein Bcl-2/Bax.