Construction of Bcl-2 shRNA expression plasmid and its inhibition on the growth of HL-60 cells

[Objective] To construct expressing vector of short hairpin RNA( shRNA ) targeting Bcl-2,and investigate the effects of Bcl-2 shRNA on the growth of HL-60 cell line. [Methods] Two of 66 base pairs oligonucleotides for short hairpin expression targeting the coding region of Bcl-2 mRNA were designed and were chemically synthesized. Double DNA sequences were obtained through annealing. Pgenesil-1 vector was linearized with BamHI and HindIII. Finally, annealed oligonucleotides were inserted into the downstream of treated Pgenesil-1 vector U6 promoter to construct RNAi plasmid. Oligonucleotide with a scrambled sequence was used as negative control. Recombinant expression vector was identified by digestion with PstI and SalI and confirmed by sequencing analysis. Bcl-2 shRNAs were transfected into HL-60 with Lipofectamine. Cytotoxic effects were measured by use of MTT method. The expression levels of Bcl-2 mRNA were assayed by RT-PCR. [Results] Identifying by enzyme cutting and sequencing showed the insertion sequence was correct. Furthermore, 66 bp had been inserted the expected site. RT-PCR assay showed that the expression levels of Bcl-2 mRNA from HL-60 cells decreased after Bcl-2 shRNAs treatment. There was no difference in Bcl-2 mRNA levels between control shRNA and untreated cells. Viable cells at 48, 72 and 96 h after treatment with Bcl-2 shRNAs were less than that after treatment with control shRNAs and untreated HL-60 cells, respectively (P 0.05). [Conclusion] Bcl-2 shRNA expression vectors have been constructed successfully, could effectively inhibit the growth of HL-60 cells.