Inhibitory Effect of RNAi on Expression of bcr/abl Oncogene in K562 Cells.

The translocation t(9;22) can be found in 95% patients with chronic myeloid leukemia (CML). The resulting hybrid gene bcr/abl codes for a fusion protein with tyrosine kinase activity which activates signal transduction pathways, leading to uncontrolled cell growth. A promising anti-gene technology-RNA interference (RNAi) reported in recent years can disrupts the expression of the targeted cellular gene in a complicated manner in a variety of organisms and cell types.

To inhibit CML bcr/abl oncogene expression with RNAi, we used chemically synthesized anti-bcr/abl small interfering RNAs( siRNAs), mismatch control siRNAs having the three point mutations to transfect the K562 cells for different time through the electroporation. EGFP plasmid was used as a positive control and the amount of fluorescently stained cells was determined by FCM. Inhibitory effects of siRNAs were demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by means of MTT assay and apoptosis was determined by Annexin V-FITC assay.

The transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. The anti-bcr/abl siRNAs reduced the bcr/abl mRNA level by 67%–72% without an obvious effect on abl mRNA levels. The K562 cells electroporated with anti-bcr/abl siRNAs contained less BCR/ABL protein than cells electroporated with mismatch control siRNAs or without any siRNAs. P210 was reduced to a very low level in Western blots, whereas the wild-type ABL protein was not influenced by the anti-bcr/abl siRNAs. Depletion of bcr/abl leading to increased apoptosis and reduction of cell viability. The flow cytometric analysis showed that the percentage of dead and apoptosic cells induced by anti-bcr/abl siRNAs for 24hrs and 48hrs were 42.10% and 53.33%, respectively, including 15.05% and 19.47% of the preliminary apoptosis cells, respectively. A significant induction of apoptosis was observed after transfection compared with untreated control(1.00%)and the mismatch control siRNAs (2.98%) (p<0.05). Anti-bcr/abl siRNAs inhibited cell growth of the K562 cells and the inhibitory rate was 47% and 56%, 24h and 48h after transfection respectively, whereas the mismatch control siRNAs had no such effect on K562 cells.

At the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study. Therefore, siRNAs may be potent tools for gene-specific modality against CML.