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

Abstract

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 lipofectAMINE^TM, 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.

Keywords

survivin / chemotherapy / hepatocarcinoma

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