The Effect And Mechanism Of Vascular Endothelial Growth Factor111b On Angiogenesis And Growth Of Ovarian Cancer

BackgroundOvarian cancer is one of the most common gynecologic malignant tumor, and itsincidence and mortality rate increased year by year. The5year survival rate of ovariancancer is only20%-30%. VEGF is generated as two families of isoforms by alternativesplicing of mRNA from8exons: VEGFxxx isoforms expressing8a exon haspro-angiogenic property, and VEGFxxxb isoforms expressing8b exon hasanti-angiogenic property. Mineur reported a new VEGFxxx family member, VEGF111,and demonstrated that it could activate VEGF-R2to promote angiogenesis. But theexistence of corresponding VEGF111b splice variants has not yet been confirmed.Objective1. To confirm the existence of VEGF111b and construct VEGF111b eukaryoticexpression system;2. To study the effect and mechanism of VEGF111b on angiogenesis of ovariancancer;3. To study the effect and mechanism of VEGF111b on growth of ovarian cancer.Methods1. RT-PCR was used to amplify VEGF111b gene from human ovarian cancerSKOV3cells treated with mitomycin C. VEGF111b gene was connected to thepcDNA3.1plasmid to construct VEGF111b eukaryotic expression vector;2. VEGF111b specific antigen peptide CRSLTRKD was used to productVEGF111b polyclonal antibody, and RT-PCR and Western blot method wereused to confirm that the VEGF111b gene and protein were expressed inovarian cancer cells;3. VEGF111b was transfected into ovarian cancer cells by LipofectaminTM2000,and screen the stably transfected cell lines by G418pressure. Western blot wasused to detecte the over-expression of VEGF111b protein in ovarian cancercells and the conditioned medium(CM);4. MTT colorimetric method was used to measure the effect on proliferation in vascular endothelial cell and ovarian cancer cells;5."Scratch" migration assay and transwell chamber were used to measure theeffect on migration in vascular endothelial cell.6. Tubule formation assay was used to measure the effect tubule forming abilityin vascular endothelial cell;7. Western blot was used to detect the mechanism and signal pathway ofVEGF111b inhibition angiogenesis;8. Colony formation assay was used to measure long-term effect on proliferationin ovarian cancer cells;9. Cell cycle analysis by flow cytometry was used to measure the effect on cellcycle in ovarian cancer cells;10. Establish ovarian cancer xenograft tumor mode of VEGF111b stabletransfection to to observe the growth of xenograft tumor.20days afterinoculation xenograft tumor was weighed; Immunohistochemical staining wasused to detect the expression of Ki67, PCNA, VEGF and CD31.Results1. The presence of VEGF111b gene was confirmed in mitomycin treated ovariancancer cells treated with mitomycin C. VEGF111b gene was amplified andconnected to the pcDNA3.1, and VEGF111b eukaryotic expression vectorwased successful constructed after double enzyme digestion and sequencinganalysis. Sequencing results are consistent with predictions;2. VEGF111b polyclonal antibody was successful producted, and confirmedVEGF111b protein were expressed in ovarian cancer cells treated withmitomycin C;3. VEGF111b stable transfection cell lines were established, and confirmedVEGF111b protein wased over-expressed in ovarian cancer cells and CM;4. VEGF111b could significantly inhibit the proliferation of vascular endothelialcells with a26±8%and38±10%decrease in proliferation at24h and48hrespectively;5."Scratch" migration experiment demonstrated that VEGF111b couldsignificantly inhibited the migration distance of vascular endothelial cells witha43%±11%and61%±13%decrease at8h and16h respectively；6. VEGF111b could significantly inhibit tubule forming ability of vascular endothelial cell;7. VEGF111b could inhibit phosphorylation of VEGF-R2and phosphorylation ofdownstream signal molecules PI3K/Akt and ERK1/2in vascular endothelialcell;8. VEGF111b could significantly inhibit the proliferation of ovarian cancer cells.VEGF111b inhibited proliferation of SKOV3with a40±7%, and50±11%decrease at48h and72h, respectively, and OVCAR3with a25±10%, and45±12%decrease respectively;9. VEGF111b could significantly inhibit colony formation of SKOV3andOVCAR3cells;10. VEGF111b could significantly induce S phase arrest in ovarian cancer cells;11. VEGF111b could inhibit phosphorylation of VEGF-R2and phosphorylation ofdownstream signal molecules PI3K/Akt and ERK1/2in ovarian cancer cells;12. VEGF111b could significantly inhibit the growth of ovarian cancer xenografttumor. In VEGF111b xenograft model, the staining of Ki67, PCNA, VEGFand CD31significantly weakened.Conclusions1. We discoveried a new VEGF splice variant-VEGF111b, and successfullyobtained VEGF111b gene sequences.2. VEGF111b polyclonal antibody was successful producted, and confirmedVEGF111b gene and protein were expressed in ovarian cancer cells treatedwith mitomycin C;3. VEGF111b inhibited the proliferation, migration and tube formation ofendothelial cells, and inhibited angiogenesis;4. VEGF111b inhibited angiogenesis by inhibiting VEGF-R2phosphorylationand its downstream signaling PI3K/Akt and ERK1/2phosphorylation;5. VEGF111b inhibited the proliferation of ovarian cancer cells, and inhibited thegrowth of tumor;6. VEGF111b induced S phase arrest in ovarian cancer cells;7. VEGF111b inhibited the proliferation of ovarian cancer cells by inhibitingVEGF-R2phosphorylation and its downstream signaling PI3K/Akt andERK1/2phosphorylation;8. VEGF111b inhibited the growth of ovarian cancer xenograft tumor. It`s associated with inhibition in angiogenesis and proliferation.