Effect of lentivirus-mediated shRNA targeting VEGFR-3 on proliferation, apoptosis and invasion of gastric cancer cells.

It has been reported that vascular endothelial growth factor receptor 3 (VEGFR-3) is highly expressed in most tumor tissues, including gastric cancer. However, the effects of VEGFR-3 knockdown on the proliferation, apoptosis and invasion of gastric cancer cells and downstream signaling molecules have not yet been well established. In the present study, four short hairpin RNA (shRNA) sequences targeting the VEGFR-3 gene (NM_002020) were designed and cloned into a lentiviral vector, pRNAT-U6.2/Lenti, to construct four recombinant lentiviral vectors. The vectors with the two highest interfering efficiencies were selected to be co-transfected with packaging vectors in HEK293T cells to assemble lentivirus particles. Results from Western blot analysis showed that the VEGFR-3 shRNA-4 lentivirus-infected group (sh#4) had the highest efficiency of gene silencing in the MKN45 cell line compared with the parental and control group. The sh#4 group significantly slowed cell proliferation, decreased the mean percentage of S-phase cells and increased the mean percentage of G1 phase cells, promoted cell apoptosis, and also significantly inhibited cell invasion of MKN45 compared with the other two groups. Furthermore, the expression of the anti-apoptotic factor Bcl-2 was significantly decreased in the sh#4 group compared to that of the other two groups. Moreover, results from qRT-PCR revealed that knockdown of VEGFR-3 with the shRNA lentiviral vector resulted in down-regulation of the downstream neural cell adhesion molecule contactin-1 (CNTN-1). In conclusion, the recombinant lentivirus particles were able to remarkably suppress VEGFR-3 expression, regulate the cell cycle, inhibit proliferation and induce apoptosis in the MKN45 cell lines.