| Chronic myelogenous leukemia (CML), an acquired abnormality that involves the hematopoietic stem cell, is a myeloproliferative disorder characterized by a cytogenetic aberration consisting of a reciprocal translocation between the chromosomes 22 and 9:t(9;22)(q34;q11). The translocation generates a novel bcr/abl fusion gene, which encodes BCR/ABL fusion protein with deregulated tyrosine kinase activity. Multiple different signaling pathways such as PI3K, RAS and STAT are activated by BCR/ABL tyrosine kinase, ultimately lead to neoplastic transformation of cell. Amounting evidences have suggested that STAT5 plays an important role in the leukemogenesis of CML, so inhibition of the expression of genes downstream of STAT5 with a transcription factor decoy has been studied in present paper, to elucidate the molecular mechanisms of constitutive activated STAT5 in the malignant transformation of CML mediated by BCR/ABL; and to explore the feasibility of targeted blockage of STAT5 as a CML therapy strategy. This study was composed of three portions described as follows: 1. Feasibility of inhibition the expressions of genes downstream of STAT5 with a decoy oligodeoxynucleotide (ODN) Transfection efficiency of decoy oligodeoxynucleotide to target cells FAM-labeled STAT5 Decoy ODN was trancfected to K562 cells mediated by cationic liposome, and slice were prepared at various time point by centrifuge. The tranfection efficiency, stability and distribution of Decoy ODN in K562 cells were observed under inverted fluorescence microscope and confocal laser microscope respectively. The results showed that Decoy ODN were transfected into K562 cells with high efficiency, the rate of fluorescence dyed cells was 95.2% at 24 hours , and at least stable for 3 days (76.0% positive cells). Stained with DAPI, confocal laser microscopy examination revealed that Decoy ODN mainly distributed within nucleus. Study of decoy oligodeoxynucleotide sequence specific binding to STAT5 Using STAT5 Decoy ODN sequence as a probe, the nuclear protein of K562 cells was extracted to perform electrophoretic mobility shift assay (EMSA), and STAT McAbs were added to perform super-shift assay at the same time. The results showed that there was a retard band in probe lane which could be competed away by excess self-probe but not by excess mutant probe; while in addition to the retarded band, there was a more retarded band in super-shift lane as well. Decoy oligodeoxynucleotide competitionally inhibition of promoter activity in K562 cells Luciferase report plasmid pGL3b-bclxp was constructed by insert the gene fragment of bcl-x promoter into pGL3b and co-transfected with Decoy ODN, Mutant ODN respectively into K562 cells. The luciferase activity was measured and normalized by protein concentration. The instant transfection results suggested that luciferase activity was significantly inhibited by Decoy ODN in K562 cells. Effects of decoy oligodeoxynucleotide on the growth of HL60 cells with non-constitutive activated STAT5 Decoy and Mutant ODN was transfected into HL60 cells respectively, cells were counted at different time point, cell growth curve was drawn and the expression of genes downstream STAT5 such as bcl-xL, cyclinD1 and c-myc were determined. The results suggested that neither Decoy nor Mutant ODN had any significant effects on the proliferation of HL60 cells and the expressions of genes downstream of STAT5.2. Reversion of the malignant phenotypes of K562 cells with the decoy oligodeoxynucleotide in vitro Cationic liposome was used to transfect Decoy ODN into K562 cells. Cells were cultured and counted to investigate the effects of Decoy ODN on K562 cell growth; MTT assay was employed to evaluate the effects of Decoy ODN on the proliferative rate of K562 cells; FCM was performed to detect cell cycle, Annexin-V staining and electron microscopy were used to determine the apoptosis of cell; RT-PCR and Western blot were employed to assay the mRNA and protein expressions of bcl-xL,cyclinD1 and c-myc; Microarray chip was used to detect the changes...
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