Design of Bcl-2 antisense oligodeoxynucleotide and study of its induction apoptosis in K562 cells

Objective:To explore the effect of antisense phosphorothioate oligodeoxynucleotide (ASODN) directed against the 5` untranslated region, the translation site and the coding region of the bcl 2 messenger RNA on apoptosis in K562 cells.Method: The computer messenger RNA secondary structure prediction software was utilized to predict the optimal and sub optimal secondary structures of human bcl 2 mRNA. Cytotoxic effects were measured by use of a cell viability assay. The expression levels of bcl 2 protein were assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by flow cytometric analysis.Result: We found that the two oligonucleotides directed against the coding region and the translation initiation of bcl 2 messenger RNA can reduce bcl 2 protein levels and K562 cells viability, and induce apoptosis. Effects of antisense oligonucleotide targeting the coding region reducing bcl 2 protein levels and apoptosis rates induced were stronger than that of antisense oligonucleotide targeting the translation initiation site.The scrambled sequence control oligonucleotides with the same base composition did not alter cell viability,bcl 2 protein expression and apoptosis rates induced in K562 cell.Conclusion: We have identified two novel antisense sequence in the translation initiation and the coding region of bcl 2 mRNA that most effectively down regulates expression of the bcl 2 proto oncogene in K562 cells. The two novel antisense sequence induce apoptosis of K562 cells in sequence specific manner.