| Background Multidrug resistance(MDR) is the major reason why chemotherapy based treatment modalities of malignant tumors may fail. MDR means malignant cells are cross resistance to many other structurally and functionally unrelated anticancer drugs once they are resisitant to a single anticancer drug. Many kinds of mechanisms contribute to MDR, such as the upregulation expression of multidrug-resistance-associated genes, the abnormal expression of apoptosis-related genes, enzyme-mediated multidrug resistance, enhanced DNA repair and micro-environment-resistant. However, the underlying mechanism conferring this MDR phenotype is the cellular overproduction of the mdr-1 gene-encoded 170-KD, membrane-spaning P-glycoprotein, a member of the super family of ABC (ATP binding cassete) transporters. Adhesion molecule CD44 is a cell surface transmembrane glycoprotein encoded by single gene, as a receptor for hyaluronic acid(HA),it involved in adhesion of extracellular matrix, angiogenesis, cell proliferation, cell differentiation and cell migration, and so on. Elevated CD44 expression was correlated with poor prognosis in hematological malignancies. It has been found that the interaction between CD44 molecules and their receptors HA may affect the malignant hematological cells survival and drug resistance. RNAi is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA), which leads to gene silencing in a sequence-specific manner. Recent years, for its high efficiency specificity and stability, RNA interference (RNAi) has become a powerful tool to knock down specific gene expression. In this study, I investigate the effect of inhibiting gene expression on the drug resistance of K562/A02, so as to provide theoretically and experimental bases for further clinical use.Objective Construction and identification of RNA interference expression vectors targeting CD44 and to investigate the effect of inhibiting gene expression on the drug resistance and biologic activity of human multidrug resistant leukemia cell line K562/A02.Methods The oligonucleotides of CD44 gene were designed referring to related date of GenBank, double-stranded DNA was derived through annealing, and inserted into pGCsilencerU6/Neo/GFP vector. The resultant recombinant plasmid pGCsiRNA-CD44 was transfected into K562/A02 cell line. Expression of CD44, MDR-1 and BCL-2 mRNA was assayed by real time RT-PCR. The Fifty-percent inhibition concentration (IC50) of doxorubicin (ADM) for K562/A02 cell line was determined by MTT method. Cell cycle was determined by flow cytometry. The morphology of apoptosis was examined by Hoechst33258 staining.Results The 4 siCD44s showed a significant inhibition of CD44 gene expression compared to the control, and siCD44-1 showed the strongest inhibitory effect. After transfected with pGCsiRNA-CD44, the expression of CD44 mRNA in K562/A02 cells was reduced by 64.1%(p<0.05),while the expression of MDR-1, BCL-2 mRNA in pGCsiRNA-CD44 transfected K562/A02 cells reduced by 25.6% and 50.8% respectively. IC50 of K562/A02 cells after transfection was decreased to (8.77±1.63)μg/ml as compared to (17.97±1.61)μg/ml of controls (p<0.01). Forty-eight hours after transfection, the ratio of K562 /A02 cells at G0/G1 increased by 10.7% and cells presented karyopyknosis, nuclear-set and apoptotic bodies.Conclusion The siRNA plasmid vector specifically targeting CD44 gene can down-regulate the expression of CD44 gene remarkably, inhibit K562/A02 cell proliferation, induce its apoptosis and effectively reverse the multidrug resistance.
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