Objective: To investigate the influence of short hairpin RNA (shRNA) expression plasmid against gene survivin (mU(6)/survivin) on survivin mRNA expression and chemosensitivity to paclitaxel in ovarian cancer cells OVCAR3.
Methods: OVCAR3 cells were transfected with plasmid pEGFPC(2) formulated with lipofectamine 2000 at different concentrations. The transfection efficiency was examined by flow cytometry. The expression of survivin mRNA of OVCAR3 cells after transfection with the plasmid mU(6)/survivin with the high efficiency ratio was observed by RT-PCR. The effect of the plasmid on the cell cycle and apoptosis was analyzed by flow cytometry. The chemosensitivities of transfected cells to paclitaxel were determined by methyl thiazolyl tetrazolium (MTT).
Results: The optimal transfection efficiency was obtained when pEGFPC(2): lipofectamine 2000 was 1: 2. Compared with the non-transfected groups, 0.81 +/- 0.05, the mRNA of survivin in OVCAR3 cells was reduced clearly after transfection with mU(6)/survivin, 0.26 +/- 0.04. shRNA reduced the expression level of survivin mRNA to 32% of non-transfected groups (P < 0.01). The apoptotic rate of survivin shRNA group reached (31.9 +/- 1.2)%, which was much higher than those of non-transfected (4.9 +/- 0.7)% and lip alone groups (5.6 +/- 0.5)% (P = 0.000). Cell cycle analysis showed that survivin shRNA induced accumulation of cells in G(0)/G(1) phase with a decrease of cells in G(2)/M phase after being cultured for 24 hours compared with non-transfected group (P < 0.01). MTT results showed that the 50% inhibiting concentration (IC(50)) of paclitaxel in non-transfected, lip alone and survivin shRNA transfected groups was (0.305 +/- 0.032), (0.157 +/- 0.031), and (0.019 +/- 0.001) micromol/L respectively. Compared with non-transfected group, shRNA increased the chemosensitivity of OVCAR3 cells to paclitaxel by 16 fold (P = 0.000).
Conclusion: Sequence specific shRNA targeting survivin can effectively suppress the expression of survivin mRNA and enhance the chemosensitivity to paclitaxel in ovarian cancer cells significantly.