Objective: To explore the inhibition effect of small hairpin RNA (shRNA) expression plasmid targeting vascular endothelial growth factor (VEGF) on VEGF expression in cultured retinoblastoma (RB) cells.
Methods: VEGF shRNA plasmid p4.1CMV-VEGF was constructed and transfected into retinoblastoma cell lines SO-RB50 and HXO-RB44. Using neomycin G418 in conjunction with gradient dilution, p4.1CMV-VEGF shRNA positive single clone of RB cells was selected and subsequently enriched. Real-time polymerase chain reaction (PCR) was applied to detect VEGF mRNA levels of RB cells. VEGF protein concentration in culture supernatants of RB cells were determined by enzyme-linked immunosorbent assay (ELISA). Plasmid p4.1CMV-Neg shRNA, which expressed shRNA lacking significant sequence identity to human and mouse genome databases, was transfected into RB cells as negative control. Cells without any treatment were used as blank controls.
Results: p4.1CMV-VEGF shRNA was constructed successfully and VEGF shRNA construct positive clone of RB cells was developed. VEGF mRNA level of SO-RB50 (HXO-RB44) cells in negative control and blank control was 5.02 (5.70) folds and 6.32 (4.86) folds greater than that in p4.1CMV-VEGF shRNA treated SO-RB50 (HXO-RB44) cells. VEGF protein concentration in culture supernatants of p4.1CMV-VEGF shRNA treated SO-RB50 cells (187.69 +/- 83.89) microg/L was significantly lower than that of negative control (822.98 +/- 187.98) microg/L and blank control (865.76 +/- 170.33) microg/L (P < 0.01). There was also significant difference of VEGF protein concentration between p4.1CMV-VEGF shRNA treated HXO-RB44 cells (162.20 +/- 66.33) microg/L and controls (764.33 +/- 164.79) microg/L in negative control and (828.22 +/- 145.94) microg/L in blank control (P < 0.01).
Conclusions: Stable transfection of VEGF shRNA expression plasmid can potently suppress VEGF expression in RB cells. RNA interference (RNAi) targeting VEGF promises to be a substantial tool for the study of the treatment of RB.