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Effects of lentivirusmediated long term bcr/abl RNAi on biologic characteristics of human leukemia cell line K562
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Abstract:
Objective: To study the effects of lentivirusmediated bcr/abl RNAi on the biological characteristics of human leukemia cell line K562. Methods:Bcr/abl RNAi lentivirus vector pNLB/AEGFP was constructed and was used to transfect K562 cells, the stable tansfectants（B/AK562）were selected. RNAi efficiency was assessed by Realtime PCR and Western blotting. Cell proliferation was detected by trypan blue staining and colony formation assay, cell differentiation was investigated by benzidine staining, PTK activity was determined by ELISA, apoptosis was observed by AOEB staining, and caspase3 and caspase9 activation were measured by chromometry. K562 cells and mock transfected EGFPK562 cells were used as controls.Results: pNLB/AEGFP stably transfected K562 cells (B/AK562) was successfully constructed. Realtime PCR and Western blotting analysis confirmed that lentivirusmediated bcr/abl RNAi downregulated bcr/abl mRNA and P210bcr/abl protein expression in K562 cells. The doubling time of B/AK562 cells was obviously longer than those of K562 cells and EGFPK562 cells (37.1 vs 20.4, 23.3 h). Furthermore, B/AK562 cells showed decreased colony formation ability, strengthened differentiation toward erythrocytes, decreased activation of PTK, increased apoptosis and enhanced caspase3 and caspase9 activation (P<0.05 or P<0.01). Conclusion: Lentivirusmediated bcr/abl RNAi can result in long time silencing of bcr/abl gene, inhibit malignant proliferation and induce differentiation and apoptosis in K562 cells.
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Supported by the National Natural Science Foundation of China (No. 30873101)
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