An Anti-cancer Study Of Antisense Oligonucleotides Targeting To Livin MRNA

A novel member of human IAPs, designated livin, was discovered recently. Its secondary structure was computed with RNAdraw and Sfold and 20 antisense oligonucleotides (20nt) targeting different regions of livin mRNA were designed, which were synthesized as phosphorothioate derivatives. The activity of antisense oligonucleotides to induce apoptosis in SK-MEL1 was measured using MTT. The results show that the oligonucleotide No. 5 could effectively inhibit cell viability by 52.6%. After transfection of oligonucleotide No. 5, microtubules of Hela cells with immnofluorescence staining and nucleus specifically stained with Hoechest33258 were photographed under laser cofocal microscope. These images showed that the cells had morphological changes, cell rounding up and cell shrinkage. It was observed also structural alternation in cytoskeleton including disorder arrangement of microtubules, disruption of microtubules, nuclear condensation, formation of multinucleated cells and aberrant mitosis. Hela cells were transfected with oligonucleotide No. 5 at concentration of 100 and 200 nmol.L-1, their mitochondrial membrane potential reduced by 6.47% and 24. 63% respectively and caspase3 activity had an increase. The level of livin mRNA of Hela transfected with oligunucleotide No. 5 reduced significantly using semi-quantitative PCR.The expression of the gene in MCF, Hela, Raji, SK-MEL1, not in B16 and HELF cells were identified using RT-PCR. CHO, HELF and Hela cells were all transfected transiently with the vector pcDNA3. 1 (+) in which livin was cloned...