Inhibition of survivin expression by small interfering RNA (siRNA) in brain tumor cell lines.

63 Survivin, a member of inhibitor of apoptosis gene (IAP) family, has been proposed as a promising target for new anticancer interventions. Here we report an application of RNA interference with small interfering RNA (siRNA) of survivin to human brain tumor cell lines. The purposes of this study are to investigate the ability and biological effects of siRNA induced inhibition of survivin, and to determine the potentiality of survivin siRNA as a new therapeutic tool in brain tumors. The survivin mRNA expression was measured by real-time RT-PCR in 7 brain tumor cell lines (Daoy, T98G, U118MG, U87MG, A172, IMR32, SH-SY5Y), and one lung adenocarcinoma cell line A549 as a positive control. Three brain tumor cell lines including Daoy (medulloblastoma), U87MG (glioblastoma cell line with wild type p53) and A172 (glioblastoma cell line with mutant p53) were transfected with siRNA oligonucleotides of survivin. Survivin mRNA and protein expressions were measured by real-time RT-PCR and Western blot analysis after 24, 48 and 72 hours of treatment with survivin siRNA, and then compared to nonsense siRNA treated cells. The effects on cytotoxicity and induction of apoptosis were assessed with MTT assay and FACS analysis using Annexin V, before and after treatment with survivin siRNA. High survivin expression was detected in all 7 brain tumor cell lines normalized with A549. After 48 hours of treatment with siRNA of survivin, survivin mRNA level was reduced by 39, 65 and 77% in U87MG, A172 and Daoy cell line, respectively, and protein expression was reduced by 30% in all 3 cell lines. Knockdown of survivin by siRNA induced 32, 32 and 38% of cell death in U87MG, A172 and Daoy cell line, respectively, after 72 hrs of treatment with siRNA of survivin. Knockdown of survivin by siRNA induced 5% of apoptosis in U87MG after 24 hrs of transfection with siRNA of survivin. The effect of siRNA was independent of p53 gene mutation. Our findings suggest that survivin may play an important role in oncogenesis of brain tumors, and survivin-specific siRNA could be a selective therapeutic tool to kill medulloblastoma and glioblastoma cells regardless of the presence or absence of wild-type p53 genes.