Design Of Antisense Drug Targeting Bcl-2 MRNA And Studies On Drug-sensitivity Of Leukemic Cells

Objective: To investigate the effect of RNA secondary structure prediction software-RNA structure on the selection of bcl-2 mRNA antisense target ; To observe the effects of the different antisense oligonucleotides on the cell viability of HL6O.. K562 , the levels of bcl-2 mRNA and protein, apoptotic cells rate and drug sensitivity; To study the differences and similarities of the antisense drugs with different structures on the biological functions of HL6O~ K562 cells; To observe the effects of the different antisense oligonucleotides on the biological fi.inctions of clinical leukemia cells. Methods:The computer messenger RNA secondary structure prediction software RNA structure was utilized to predict the optimal and sub-optimal secondary structures of human bcl-2 mRNA. A BLAST search of a database containing all sequences in GeneBank reveal no homology of the sequences to the other human genes; In order to minimize the risk of self-hybridization or hairpin formation, the program Quickfold was used to verifying that the sequences selected do not fold back on themselves. The Lalign program was used to ensure that the oligonucleotide sequences do not dimerize. Cytotoxic effects were measured by use of a cell viability assay; Apoptosis was detected by morphological observation and DNA gel electrophoresis; Flow cytometric analysis of DNA fragmentation was also performed. The expression levels of bel-2 messenger RNA and protein were assayed by reverse transcriptase polymerase chain reaction arid immunofluorescence using fluoresce isothiocyanate label respectively. Results: We found that the two oligonucleotides directed against the coding region 6 I and the translation initiation of bcl-2 messenger RNA in the five I 8-mer phosphorothioates directed against the 5?untranslated region, the translation site and the coding region of the bcl-2 messenger RNA can reduce HL6OS K562 cells viability; the oligonucleotides 1 directed against the coding region of the bcl-2 messenger RNA was more cytototic than the olgonucleotides 2 that targeted the translation initiation. The results indicated that computer aided antisense drug design basing on secondary structure prediction was helpful to obtain antisense oligonucleotide sequence with better effects. Consistent with the results on cell viability, oligonucleotides caused a significant decrease in expression of the bcl-2 peotein and the effects were dose and time dependent. The antisense oligonucleotides directed against the coding region and the translation initiation of bcl-2 messenger RINA reduced messenger RNA levels. In contrast to antisense oligonucleotide 2 targeting the translation initiation site of the bcl-2 messenger RNA, effects of antisense oligonucleotide I targeting the coding region reducing bcl-2 protein levels and mRNA levels were stronger than that of antisense ? oligonucleotide 2. The scrambled sequence control oligonucleotides with the same base composition did not alter leukemia cell viability~ bcl-2 protein expression and bcl-2 messenger RNA levels even when they were tested at higher concentrations. These results provide strong evidence for a sequence-specific mechanism of action. Antisense oligonucleotide 1 and 2 increase the sensi...