Design and synthesis of efficient bcl-2 siRNA and it's interference role to HL-60 cells in leukemia

Objective To study the interference role of efficient bcl-2 siRNA to HL-60 cells in leukemia. Methods Beginning with the AUG start codon, scan the length of bcl-2 mRNA for AA sequences. Record the AA and downstream 19 nucleotides and compare the sequence to the appropriate genome database to eliminate any sequences with significant homology to other genes. In this study, we selected three target sequences for testing and controlled the GC content between 30 % ~ 65 %, because early experiments suggest that target sequences with a low GC content may be more susceptible to siRNA-induced degradation. bcl-2 siRNA was synthesized in vitro transcription with silencer siRNA construction kit. Synthesized siRNA was transfected into HL-60 cells with lipid siPORT transfection Kit. Forty-eight hours after transfection, we collected cells and detected the bcl-2 protein expression by flow cytometer. We selected the mostly efficient siRNA to do subsequently experiment according to the flow cytometerys result. We used RT-PCR and immunofluorescence to determine the expression level of bcl-2 mRNA and protein. Results bcl-2 siRNA1 specifically reduced bcl-2 expression on the level of mRNA and protein in HL-60 cells and induced cell apoptosis. There was no difference on the effect of other groups compared with the control groups. And GAPDH siRNA also only reduced the GAPDH gene expression while had no influence to bcl-2 gene. Conclusion We adopted bioinformatic technique to design effective target siRNA; Effective bcl-2 siRNA could specifically degrade bcl-2 protein expression by degraded bcl-2 mRNA level.