Inhibition of the VEGF expression and cell growth in hepatocellular carcinoma by blocking HIF-1alpha and Smad3 binding site in VEGF promoter.
In order to investigate the inhibitory effects on the vascular endothelial growth factor (VEGF) expression and cell growth in hapatocellular carcinoma (HCC) by blocking HIF-1alpha and Smad3 binding site in the VEGF promoter, antisense oligodeoxynucleotides (ASODN) were designed to block HIF-1alpha and Smad3 binding site in the VEGF promoter. Different concentrations of ASODN and ODN were transfected into HCC cells respectively. The expression of VEGF mRNA and protein was detected by SABC, Western blot and RT-PCR techniques and the inhibitory effects on the expression of VEGF and cell growth of the HCC cells stimulated by the supernatants were determined by using MTT method. Immunohistochestry revealed that after co-inoculation of hepatocellular carcinoma cells with different concentrations of ODN and ASODN for 48 h, there was no significant difference in the expression of VEGF protein between ODN group and control group (P > 0.05), but there was significant difference between ASODN group and control group (P < 0.05). At a concentration of 10 micromol/L ASODN, the difference was very significant (P < 0.01). Western blot and RT-PCR revealed that, after treatment for 48 h at a concentration of 10 micromol/L. the integral gray levels and RNA odds were 59743.2 +/- 10412.5 and 0.783 +/- 0.032 in ODN group, and 38694.5 +/- 10925.1 and 0.468 +/- 0.015 in ASODN group, respectively, with the difference being very significant (P < 0.01). Antisense ODN could inhibit the growth of HCC cells in a concentration-dependent manner. It was concluded that anti-gene technique of aiming at HIF-1alpha action site in the VEGF promoter could suppress the VEGF expression and inhibit HCC cell growth, and it is promising that anti-gene technique works as a new gene therapeutic tool for anti-angiogenesis of HCC.
Ding
Lei
L
Department of Hepatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Chen
Xiaoping
X
Jing
Kai
K
Wang
Haiping
H
Zhang
Wanguang
W
D001665:Binding Sites / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051900:Smad3 Protein / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
D051793:Hypoxia-Inducible Factor 1; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D042461:Vascular Endothelial Growth Factor A