Downregulation of survivin by RNAi inhibits growth of human gastric carcinoma cells

Abstract

Aim: To investigate the inhibitory effect of a specific small survivin interfering RNA (siRNA) on cell proliferation and the expression of survivin in human gastric carcinoma cell line SGC-7901.

Methods: To knockdown survivin expression, a small interfering RNA targeting against survivin was synthesized and transfected into SGC-7901 cells with lipofectamine 2000. The downregulation of survivin expression at both mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Cell proliferation inhibition rates were determined by methyl thiazolyl tetrazolium (MTT) assay. The effect of survivin siRNA on cell cycle distribution and cell apoptosis was determined by flow cytometry (FCM).

Results: RNA interference could efficiently suppress the survivin expression in SGC-7901 cells. At 48 h after transfection, the expression inhibition rate was 44.52% at mRNA level detected by RT-PCR and 40.17% at protein level by Western blot analysis. Downregulation of survivin resulted in significant inhibition of tumor cell growth in vitro. The cell proliferation inhibition rates at 24, 48 and 72 h after survivin siRNA and non-silencing siRNA transfection, were 34.06%, 47.61% and 40.36%, respectively. The apoptosis rate was 3.56% and the number of cells was increased in G(0)/G(1) phase from 38.2% to 88.6%, and decreased in S and G(2)/M phase at 48 h after transfection.

Conclusion: Downregulation of survivin results in significant inhibition of tumor growth in vitro. The inhibition of survivin expression can induce apoptosis of SGC-7901 cells. The use of survivin siRNA deserves further investigation as a novel approach to cancer therapy.

Figure 1

Expression of survivin mRNA in different groups of SGC-7901 cells. A: Survivin mRNA expression in RT-PCR assay; B: Survivin mRNA expression in SGC-7901. 1: treated with survivin siRNA; 2: treated with non-silencing siRNA; 3: untreated. Data are expressed as mean ± SD of three experiments, survivin siRNA group vs non-silencing siRNA group, ^bP < 0.01 ; survivin siRNA group vs untreated group, ^bP < 0.01.

Figure 2

Western blot assay of survivin protein expression in SGC-7901 cells. A: Survivin protein expression in Western blot assay; B: Survivin protein expression in SGC-7901.1: untreated; 2: non-silencing siRNA; 3: survivin siRNA. Data are expressed as mean ± SD of three experiments, survivin siRNA group vs non-silencing siRNA group, ^bP < 0.01; survivin siRNA group vs untreated group, ^bP < 0.01.

Figure 3

Effects on cell viability after non-silencing siRNA and survivin siRNA treatment. 1: untreated; 2: non-silencing siRNA; 3: survivin siRNA. Data are expressed as mean ± SD, survivin siRNA group vs non-silencing siRNA group, ^bP < 0.01; survivin siRNA group vs untreated group, ^bP < 0.01.