Inhibitory effect of antisense vascular endothelial growth factor 165 eukaryotic expression vector on proliferation of hepatocellular carcinoma cells

摘要

AIM: To construct antisense VEGF165 eukaryotic expression vector PCDNA3-as-VEGF165 and to study its expression and effect on the proliferation of hepatocarcinoma SMMC-7721 cells.METHODS: VEGF165 cDNA was inserted into polylinker sites of eukaryotic expression vector PCDNA3 to construct PCDNA3-as-VEGF165. Then the vector was transferred into human hepatocarcinoma cell strain SMMC-7721 with cation lipofectamine 2000 mediated methods to evaluate the expression of VEGF protein and the inhibitory effect on the proliferation of hepatocarcinoma SMMC-7721 cells.RESULTS: The detection indicated the presence of VEGF cDNA in normally cultured SMMC-7721 cells by PCR. VEGF mRNA expression was notably decreased in SMMC-7721 cells by RT-PCR after PCDNA3-as-VEGF165 transfection. The expression of VEGF protein was dramatically inhibited (142.01 ± 7.95 vs 1 625.52 ± 64.46 pg·ml^-1, P < 0.01) 2 days after transfection, which correlated with the dose of PCDNA3-as-VEGF165 gene. VEGF protein was most expressed in PCDNA3 transferred SMMC-7721 cells but few in PCDNA3-as-VEGF165 transferred cells by immunohistochemical staining. The apoptotic rate of hepatocarcinoma SMMC-7721 cells was significantly promoted (17.98% ± 0.86% vs 4.86% ± 0.27%, P < 0.01) and the survival rate was notably decreased (80.99% ± 3.20% vs 93.52% ± 3.93%, P < 0.05) due to antisense VEGF165 by flow cytometry (FCM). The transfection of antisense VEGF165 gene resulted in the inhibitory effect on the proliferation of hepatocarcinoma cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and the death of all hepatocarcinoma cells on day 6 after transfection.CONCLUSION: It is confirmed that antisense VEGF₁₆₅ can inhibit the expression of VEGF protein, interfere with the proliferation and induce the apoptosis of hepatocarcinoma cells in our study. Antisense VEGF₁₆₅ gene therapy may play an important role in the treatment of human hepatocarcinoma.