In vitro application of RNA interference to silence livin gene expression to induce apoptosis in leukemia cells

J. Lv, Z.-P. Qin, M.-F. Zheng, Z.-C. Chen, Z. Wang

LinYi Tumor Hospital, Linyi, China. wangzhen515_14@163.com

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• Hematology

OBJECTIVE: To search for new targets and novel methods of anti-leukemia treatment and to discuss the mechanism of silencing the livin gene using small RNA interference technology to induce apoptosis in the K562 leukemia cell line.

METHODS: We designed and synthesized livin-specific small interference RNA (siRNA). Transfected K562 cells were cultured. Reverse-transcription polymer chain reaction (RT-PCR) was used to detect livin mRNA expression. Protein expression for livin was detected using Western blotting. A non-transfected group was used as a control. Meanwhile, vectors carrying enhanced green fluorescent protein (EGFP) were transfected as a positive control and flow cytometry was used to determine the transfection efficiency by detecting green fluorescence. The rate of apoptosis was determined using the annexin V and propidium iodide double-staining method. ELISA was used to determine the activity of Caspase-3.

RESULTS: The transfection efficiency of electroporation was as high as 50%. The siRNA sequences could knockdown livin gene expression at both the mRNA and protein levels. Apoptosis rate of the cells was 27.41 ± 2.30% 48 h after transfection with specific siRNA. This was significantly higher than that of the control group (9.63 ± 0.89%, p < 0.05). The 48-h apoptosis rate of the combined effect group VP-16 (5 µmol/L) and transfection rate was 45.1 ± 4.40%, which was significantly elevated (p < 0.05) compared with the groups treated with only VP-16 or by transfection.

CONCLUSIONS: Caspase-3 activity in cells transfected with siRNA was significantly elevated compared to the cells in the non-transfected groups (p < 0.05).

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