An antisense plasmid targeting survivin expression induces apoptosis and sensitizes hepatocarcinoma cells to chemotherapy

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Abstract
Summary To explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cellsin vitro. Survivin mRNA structure region was amplified by RT-PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINETM, 2000 (FL2000), with low concentration of 5-fluorouracil (5-Fu) added. Survivin protein was detected by westernblot, the growth activity was measured by MTT, and apoptosis was detected by Flow cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase-3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5-Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase-3 activity were increased in antisense survivin transfected + 5-Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.