Effects and mechanism of downregulation of survivin expression by RNA interference on proliferation and apoptosis of lung cancer cells

Abstract

The survivin protein, a member of the inhibitors of apoptosis (IAP) family, has gained popularity as a therapeutic target for cancer due to its selective expression in tumor cells and its significant involvement in tumor cell viability. The aim of this study was to investigate the effect of the survivin-small interfering RNA (siRNA) plasmid on survivin expression in the human lung cancer cell line, A549, and to observe its effects on apoptosis and proliferation of A549 cells. A549 human lung cancer cells were transfected with survivin-targeting siRNA. The downregulation of survivin expression was determined by real-time polymerase chain reaction and western blotting. The proliferation of A549 cells was determined by MTT assay. The apoptotic rate and cell cycle distribution were analyzed by flow cytometry (FCM). Caspase-9 activity was also detected to study the apoptosis of lung cancer cells induced by siRNA against survivin. The sequence-specific siRNA efficiently and specifically downregulated the expression of survivin at both the mRNA and protein levels. Downregulation of survivin expression dramatically suppressed the proliferation of A549 cells and arrested the cells at the G (1)/G (0) phase. Caspase-9 activity was significantly increased in A549 cells transfected with siRNA against survivin. In this study, we found that survivin-specific siRNA can efficiently suppress the expression of survivin, increase apoptosis and inhibit A549 cell proliferation. Our findings further indicate the possibility that the antitumor effects of survivin-siRNA are mediated through the activation of caspase-9.

Figure 1

(A) KD1 shows the clearly inhibition of the expression of survivin as detected by western blot analysis; (B) Evaluation of transfection efficiencies showed the high transfection efficiency 24 h after survivin-siRNA transfection.

Figure 2

siRNA-survivin transfectants showed a remarkably decreased expression of survivin in A549 cells.

Figure 3

The proliferative activity of siRNA for the inhibition of survivin expression in A459 cells on days 1–5 assessed by the MTS assay.

Figure 4

Analysis of the cell cycle showed that following the downregulation of survivin expression, the proportion of cells in the G0/G1 phase increased whereas the proportion of cells in the S phase decreased.

Figure 5

Expression of caspase-9 in A549 cells shown by western blotting and analyzing the gradation. (A) Survivin inhibition increased caspase-9 expression in A549 carcinoma cells. (B) The gradation ratio of caspase-9.