The Effects Of STAT5 Decoy ODNS ON K562

Objective: CML is characterized by the reciprocal chromosomal translocation 9:22, which generates the Philadephia chromosome. This translocation generates a novel fusion gene, BCR/ABL, that encodes BCR/ABL fusion protein , with a hyperactive tyrosine kinase. Several studies have identified signaling pathways activated by BCR/ABL, including Ras/MAPK, PI3K, STAT5, and so on. Especialy, STAT5 is critical for CML transformation. Transcription factor decoy is a new techniques for gene therapy and in the study of transcriptional regulation. Nucleic acid molecules with high affinity for target transcription factor can be introduced into cells as decoy cis-elements to bind these facors and alter gene expression. When delivered into the nucleus of a target cell, the decoy ODNs can bind to free transcription factor protein and block its interaction with the promoter region of the target genes under its transcriptional regulation. Here, we try to use transcription factor decoy to inhibite STAT5 signaling pathway of K562, then observe the effect of Decoy ODNs in the proliferation, apoptosis and modulation of STAT5-mediated gene expression. This may help us to understand the mechanism of STAT5 in K562 more clearly and explore the STAT5 Decoy ODNs for therapy CML.Methods: Fist, the STAT5 Decoy ODNs, a double-stranded phosphorothioate 21mer, that exhibits a high sequence-specific binding affinity to the transcription factor STAT5 was synthesized. Cationic liposomes were used to transfect double-stranded Decoy ODNs into K562 cells. We make cell count experiments to determine the effects of the STAT5 Decoy ODNs on K562 cell growth. The MTT assay evaluated the effects of STAT5 Decy ODNs on the proliferative rate of K562 cells. The cell cycle assay and Annexin-V staining method to calculated apoptic cells by FCM. Then, we use RT-PCR method to observe the effects of STAT5 Decoy ODNs on the expression of bcl-xL,cyclin D1 and c-myc mRNA. Western blotting technique were used to determine the effect of stat5 Decoy ODNs on the expression of bcl-xL,cyclin D1 and c-myc proteins.Result: 1. The stat5 Decoy ODNs could inhibit the proliferation of K562 cells, and the inhibite increased with the dose increasing. 5μΜ Decoy ODNs could not inhibite the proliferation of K562 cells well, but 15μΜ and 25μΜ could . The result of MTT assay is in accordance with this. The proliferative inhibition rate of 5μΜ, 15μΜ and 25μΜ Decoy ODNs were 15%, 51% and 82%. But the proliferative inhibition of Mutant ODNs is very limited.2. The cell cycle assays of Decoy ODNs on K562 cells indicated that the percentage of S phase cells decreased from48.71±5.42％ to 31.94±2.35%(P<0.05), while the percentage of M0/M1 phase cells increased from 40.83±5.20％ to 61.39±2.18％(P<0.05). The untreated K562 cells and treated with Mutant ODNs did not effect their cell cycles significantly.3. After treated with Decoy ODNs 24h, FCM-Annexin-V/PI duel staining method determined the percentage of early apoptotic cells is 14.57±1.08%, while the cells and treated Mutant ODNs is 4±2.02%. Electron microscope also determined the microcosmine changes of apoptotic cells.4. RT-PCR tests indicated that STAT5 Decoy ODNs had decreased the mRNA expression of bcl-xL, cyclin D1 and c-myc. Western blotting tests also determined decrease of protein expression of bcl-xL, cyclin D1 and c-myc.Conclusion: First, STAT5 Decoy ODNs could inhibite the proliferation of K562, and the inhibite rate is dose-dependent. Second, Decoy ODNs altered the cell cycle progress and induced K562 apoptosis. Last, Decoy ODNs decreased the expression of of bcl-xL, cyclin D1 and c-myc both the mRNA and the protein, and it is maybe the cause of Decoy ODNs effecting the cell cycle and apoptosis.