Effects of Her-2/neu siRNA-mediated gene silencing on cell cycle and apoptosis of lung adenocarcinoma cells

Abstract

To investigate the effect of synthesized Her-2/neu specific siRNA on the cell cycle and apoptosis of Her-2/neu upregulating human lung adenocarcinoma cells. Human lung cancer cells of the line calu-3 were cultured and divided into 4 groups: untreated control group, blank vector group transfected with blank vector, non-specific siRNA group transfected with unrelated siRNA, and Her2/neu siRNA group transfected with Her2/neu siRNA. RT-PCR was used to examine the Her-2/neu and cyclin D(1) mRNA expression. Flow cytometry was used to examine the Her-2/neu protein expression and cell cycle. The apoptosis rate was analyzed by using annexin V-FITC kit. The vascular endothelial growth factor (VEGF) level in the culture supernatant was detected by ELISA. Twenty-four hours after transfection, the expressions of Her-2/neu mRNA and cyclin D1 mRNA in the Her2/neu siRNA group decreased remarkably, both significantly lower than those in the other 3 groups. Forty-eight hours after transfection, the expression rate of Her-2/neu protein was 25.0% +/- 1.6% in the calu-3 cells transfected with Her-2/neu siRNA, significantly lower than in the control group, blank vector group, and non-specific siRNA group (98.2% +/- 2.2%, 95.7% +/- 2.0%, and 94.8% +/- 1.6% respectively, all P < 0.01); the proportion of the cells in G(0)/G(1) stage increased and those in the S stage decreased in the celu-3 cells transfected with Her-2/neu siRNA, and the proportions of the cells in G(0)/G(1) stage and Stage did not significantly change (F = 6.1, P < 0.01); the apoptotic rate of the Her2/neu siRNA group was 25.1% +/- 1.2%, significantly higher than those of the other 3 groups (4.8% +/- 0.5%, 8.6% +/- 0.9%, and 10.3% +/- 0.3% respectively, all P < 0.01); the caspase-3 activity ratio of the Her2/neu siRNA group was 134.6% +/- 4.5%, significantly higher than those in the blank vector group and non-specific siRNA group (105.0% +/- 2.5% and 112.0% +/- 2.8% respectively, both P < 0.01), and the VEGF level in the supernatant of the Her2/neu siRNA group was 176 pg/ml +/- 6 pg/ml, significantly lower than those of the control group, blank vector group, and non-specific siRNA group (476 pg/ml +/- 13 pg/ml, 426 pg/ml +/- 9 pg/ml, and 406 pg/ml +/- 9 pg/ml respectively, all P < 0.01). Chemically synthesized Specific Her-2/neu targeting siRNA effectively inhibits Her-2/neu expression and leads to the decline of cyclin D(1) and VEGF levels and activation of caspase-3 pathway thus arresting the cell cycle at G(0)/G(1) stage and enhancing cell apoptosis.