[Vascular endothelial growth factor shRNA mediated by pEGFP-H1 vector plasmid effectively inhibits glioma proliferation: an experimental study]

Abstract

Objective: To investigate the inhibitory effects of RNA silencing via plasmid-mediated vascular endothelial growth factor (VEGF) shRNA on proliferation of glioma cells in vitro and in vivo.

Methods: pEGFP-H1/VEGF vector plasmid containing enhanced green fluorescent protein (EGFP) gene and expressing VEGF shRNA was constructed. The EGFP expression was detected by fluorescent microscopy and flow cytometry. Glioma cells of the line U251 were cultured and divided into 3 groups to be transfected with blank vector, pEGFP/H1plasmid, and pEGFP-H1/VEGF respectively. RT-PCR was used to detect the mRNA expression of VEGF in the supernatants of the culture media. The protein expression of VEGF in the supernatant was detected by ELISA. The cell growth was observed with MTT method. Fifteen nude rats were randomly divided into 3 equal groups to be transplanted with U251 cells and pEGFP-H1, U251 cells and pEGFP-H1/VEGF, and U251 cells only as blank control group. The growth of glioma was observed every day. 30 days after the rats were killed and the tumors were taken out to be examined.

Results: Twenty-four hours after transplantation, fluorescence microscopy showed great numbers of U251 cells that expressed green fluorescence in both the pEGFP-H1 group and pEGFP-H1/VEGF group. Flow cytometry showed that the rates of green fluorescent protein positive cells were 68.37% and 65.29% in these 2 groups (P > 0.05). MTT method showed no significant difference in the effect on cell growth among the 3 groups (all P > 0.05). PCR showed that the VEGF mRNA expression was significantly inhibited in the pEGFP-H1/VEGF group in comparison with those in the blank control group and the group transfected with pEGFP-H1 (both P < 0.05). The concentration of VEGF protein of the U251 cells transfected with pEGFP-H1/VEGF was 530 ng/L +/- 118 ng/L, significantly lower than those of the control group (2571 ng/L +/- 572 ng/L) and the group transfected with pEGFP-H1 (2402 ng/L +/- 310 ng/L, by 77.9%) (both P < 0.01). Tumor could be touched 13 days after transplantation in the rats of pEGFP-H1/VEGF group, markedly later than in the other 2 groups. Thirty days after, the weight and volume of tumor were 0.5 g +/- 0.4 g and 401 mm(3) +/- 272 mm(3) respectively in the rats of pEGFP-H1/VEGF group, both significantly lower than those of the control group and pEGFP-H1 group (1.7 g +/- 0.4 g and 1573 mm(3) +/- 330 mm(3); and 1.5 g +/- 0.7 g and 1430 mm(3) +/- 382 mm(3)) (all P < 0.01).

Conclusion: The successfully constructed shRNA targeting at VEGF efficiently decreases the VEGF expression of the glioma cells in vitro and suppresses the growth of glioma cells in vivo.