Objective： To investigate the effects of small hairpin RNA（shRNA） targeting Bcl-2 on the growth of NB4 cell line. Methods： Two of pairs oligonucleotides for short hairpin expression targeting the coding region of ...Objective： To investigate the effects of small hairpin RNA（shRNA） targeting Bcl-2 on the growth of NB4 cell line. Methods： Two of pairs oligonucleotides for short hairpin expression targeting the coding region of Bcl-2 mRNA were designed and chemically synthesized. Annealed oligonucleotides were inserted into Pgenesil-1 vector downstream of U6 promoter to construct RNAi plasmid. Oligonucleotide with a scrambled sequence was used as a negative control. Recombinant expression vector was identified by enzyme cutting and sequencing. Bcl-2 shRNAs were transfected into NB4 cell with Lipofectamine 2000. Western-Blot of Bcl-2 protein expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a MTT assay. Results： Enzyme cutting and sequencing showed that the insertion sequence was correct. Western-Blot assay showed that the expression level of Bcl-2 protein in NB4 cells decreased after Bcl-2 shRNAs treatment. There was no difference in Bcl-2 protein levels between control shRNA and untreated cells. Viable cells at 72 and 96 h after treatment with Bcl-2 shRNAs were less than that after treatment with control shRNAs and untreated NB4 cells, respectively （P〈0.05）. Control shRNA had no significant effect on the growth of the cells. Conclusion： Bcl-2 shRNA could effectively inhibit the growth of NB4 cells. Bcl-2 shRNA might be an effective anti-leukemia candidate.展开更多
基金This work was supported by a grant from the Natural Science Program Foundation of Guangdong Province (No.04010446)
文摘Objective： To investigate the effects of small hairpin RNA（shRNA） targeting Bcl-2 on the growth of NB4 cell line. Methods： Two of pairs oligonucleotides for short hairpin expression targeting the coding region of Bcl-2 mRNA were designed and chemically synthesized. Annealed oligonucleotides were inserted into Pgenesil-1 vector downstream of U6 promoter to construct RNAi plasmid. Oligonucleotide with a scrambled sequence was used as a negative control. Recombinant expression vector was identified by enzyme cutting and sequencing. Bcl-2 shRNAs were transfected into NB4 cell with Lipofectamine 2000. Western-Blot of Bcl-2 protein expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a MTT assay. Results： Enzyme cutting and sequencing showed that the insertion sequence was correct. Western-Blot assay showed that the expression level of Bcl-2 protein in NB4 cells decreased after Bcl-2 shRNAs treatment. There was no difference in Bcl-2 protein levels between control shRNA and untreated cells. Viable cells at 72 and 96 h after treatment with Bcl-2 shRNAs were less than that after treatment with control shRNAs and untreated NB4 cells, respectively （P〈0.05）. Control shRNA had no significant effect on the growth of the cells. Conclusion： Bcl-2 shRNA could effectively inhibit the growth of NB4 cells. Bcl-2 shRNA might be an effective anti-leukemia candidate.
