Survivin small interfering RNA suppresses glioblastoma growth by inducing cellular apoptosis

Abstract

A survivin small interfering RNA sequence specific for a human and mouse homogenous sequence was constructed. Survivin small interfering RNA could significantly inhibit glioma cell proliferation and induce apoptosis when it was transfected into either a human glioma cell line U251 or rat glioma C6 cells in vitro. In addition, treatment of rat orthotopic glioma models with survivin small interfering demonstrated the inhibition of glioma growth in vivo. Our experimental findings suggest that the use of RNA interference techniques to target the survivin sequence may be useful in the treatment of glioma.

Keywords: RNA interference; apoptosis; glioblastoma; survivin.

Figure 1

Representative fluoromicrographs of apoptosis detected by acridine orange/ethidium bromide assay in U251 and C6 cells (× 200). White arrows indicate early stage apoptotic cell and blue arrows indicate late stage apoptotic cell. siRNA: Small interfering RNA.

Figure 2

Western blot analysis of proteins expression in U251 cells. (A) Survivin and its regulatory proteins expression in U251 cells in the untreated, control vector, and survivin siRNA groups at 48 hours after transfection. (B) Amounts of survivin and its regulatory proteins (Bcl-2, cyclin D1, c-Myc and caspase-3) in U251 cells transfected with survivin siRNAs. Data are expressed as mean ± SD in three independent experiments. ^aP < 0.05, vs. untreated and control vector groups (two-tailed t-test). siRNA: Small interfering RNA.

Figure 3

Photomicrographs of rat brains treated with survivin siRNA resulting in significant inhibition of tumor growth through computed tomography scan. Red arrows represent glioblastoma. The rat C6 glioma treated with phosphate-buffered saline (A), scrambled siRNA (B), and survivin siRNA (C) by day 21. Compared with untreated and control vector groups, survivin siRNA treatment resulted in obvious growth inhibition of giloma cells. siRNA: Small interfering RNA.

Figure 4

Western blot analysis of survivin protein level from C6 xenograft. (A) Survivin protein expression level in the untreated, control vector, and survivin siRNA groups from xenograft. (B) The relative amounts of survivin after treatment, data are expressed as mean ± SD in three independent experiments. ^aP < 0.05, vs. untreated and control vector groups (two-tailed t test). siRNA: Small interfering RNA.

Figure 5

Hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and Ki67 immunohistochemistry analysis for rat glioma (× 100). (A, D, G) In the untreated groups, the tumors grow very strong and apoptosis cells are little. (B, E, H) In control vector group, apoptotic cells are also little, Ki67 positive cells are everywhere. (C, F, I) In survivin small interfering RNA group, apoptotic cells increase significantly while Ki67 immuno-positive cells are reduced greatly. Positive cells: brown.