Knockdown of VEGF-A expression in neuroblastoma cell line SH-SY5Y by RNA interference

Objective To construct the short hairpin RNA(shRNA) vector of VEGF-A and down-regulate the expression of VEGF-A through RNA interference in neuroblastoma cell line SH-SY5Y. Methods Two pairs of oligonucleotide sequences were designed and synthesized with human VEGF-A cDNA. The annealed oligonucleotide fragments were subcloned into plasmids psc001 psc002. After being identified by PCR, sequencing and selection by Southern Blot, the recombinant plasmids were transfected into SH-SY5Y cells. Doubly tagged by Annexin V and PI, the transfected cells were sorted by FCMS for apoptosis after 48 hours. VEGF-A expression in the transfected cells was detected by ELISA. Results PCR and sequencing showed that the oligonueleotide fragrnents were correctly in-serted into plasmids psc001, psc002 respectively. VEGF -A expression in the transfected cells was knocked down significantly. Conclusions The shRNA expression vector of VEGF-A was successfully constructed and could significantly down-regulate VEGF-A expression in SH-SY5Y cells without lead-ing to apoptosis in the transfected cells. Key words: Vascular endothelial growth factor;  RNA interference;  Neuroblastoma;  Apoptosis.