| RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Small interfering RNA or short interfering RNA (siRNA), a kind of 21- and 22- nucleoited (nt) double-stranded RNA synthesized in vitro, has the ability to trigger RNAi. According to the recent reports, siRNA duplexes can specifically and effectively suppress expression of endogenous and heterologous genes in different mammalalian cells, avoiding the nonspecific degradation of gene and death of cells mediated by long-stranded dsRNA. There are several vitues of siRNA: the high specification of the recognization with target mRNA; the low concentration for triggering RNAi and the recycled regeneration; the resistance to nuclease. Compared with single-stranded antisense nucleotide, the effect of gene degradation mediated by siRNA is more effective and specific, while the necessary concentration is lower. siRNA duplex synthesized hi vitro are composed of 21~25nts sense and 21-25 nts antisense strands with 3'-OH and 5'-PO3, paired in a manner to have a 2-nt 3' overhang.Multi-drug resistance (MDR) is one of the most important reasons on therapy failure in leukemia. It's necessary to find an effective method to reversal MDR. Lung resistance-related protein (LRP) is a kind of MDR related glycoprotein, which induces drug accumulation deficiency in cells and leads to MDR in many kinds of tumor cells. It has been proved that over-expression of LRP is one of the most important mechanisms for MDR in leukemia cells. In acute myeloid leukemia of adults and in acute leukemia of children, the expression of LRP in leukemia cells is an independent prognosis factor and its over-expression predicts insensitive with chemical therapy drug, which induces lower remission rate, longer time for remission and easier relapse; there are more cases with over-expression of LRP in leukemia cells in failed and recurrent leukemia patients than that in chemical drug untreated or sensitive leukemia patients. Because the mechanism of drug-resistance is ATP-independed, drugs used to reverse MDR induced by P-glycoprotein, such as verapamil and cyclo-C, have no effect on the reversal of LRP-induced MDR. By now, there are a few researches on the reversal of LRP-induced MDR in tumor cells and no one on that in leukemia cells. It isimportant to find an effective method to repress over-expression of LRP in leukemia cells for the reversal of LRP -induced MDR.We transfected LRP gene-specific siRNA designed by ourselves into the MDR leukemia cells to degrade LRP mRNA and inhibit the protein expression, hi order to find an effective method to reversal MDR in leukemia cells mediated by LRP.In the study, we performed the following experiments:(1)The cells model of K562 cells line in vitro which over-expressed LRP induced by NaB was constructed, Compared with the untreated K562 cells, LRP mRNA level and the protein, expression in K562 cells treated with NaB were increased significantly; the daunorubicin (DNR) accumulation in the cells was decreased; the distribution of adriamycin (ADM) was decreaded in the nuclei and was increased in the cytoplasm.(2)The full-length cDNA of LRP produced by polymerase chain reaction (PCR) was cloned into site between BamHI and Hindlll of plasmid pcDNA3/LRP. DNA sequencing showed the sequence of LRP gene in pcDNA3/LRP was the same as the sequence of LRP cDNA in human gene bank. LRP mRNA and the protein expressed hi K562 cells transfected with pcDNA3/LRP.(3)The LRP sequence-specific siRNA was designed by the authors according to the principle of siRNA. No 100% homologous mRNA of other genes was found in Blast. The result of computer-assistant analysis for the siRNA showed the LRP specific siRNA designed by the authors might be useful for RNAi.(4)LRP siRNA and pcDNA3/LRP were cotransfected into K562 cells with lipofectamine 2000 to detect the effectiveness of siRNA-LRP on the LRP mRNA degradation...
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