[Targeted inhibition of vascular endothelial growth factor (VEGF) expression in human osteosarcoma cell line by antisense VEGF165 cDNA promoted by hypoxia reaction element].
OBJECTIVE
Utilizing the hypoxia inducible factor 1/hypoxia reaction element (HIF-1/ HRE) gene regulation system to construct antisense vascular endothelial growth factor (VEGF165) cDNA eukaryotic expression vector promoted by HRE, and investigate its targeted inhibiting VEGF expression of osteosarcoma cells in hypoxia environment.


METHODS
Eukaryotic expression plasmid with HRE promoter was constructed containing luciferase reporter gene and antisense VEGF165 cDNA by using PCR and recombinant DNA techniques. The recombinant vectors were transfected into osteosarcoma cells with lipofectin method. Hypoxia-inducible reporter gene expression was determined by liquid scintillation analyzer and the expression of VEGF protein was detected by ELISA method.


RESULTS
The eukaryotic expression plasmid containing antisense VEGF165 and luciferase promoted by HRE was constructed successfully. After being transferred into MG63 cells, luciferase expression was increased 3.5 x 10(2) times and VEGF protein expression decreased 45% under hypoxia condition.


CONCLUSIONS
Antisense VEGF165 cDNA expression, efficiently realized by HRE promoter under hypoxia condition, provides an experimental basis for targeted antiangiogenesis of tumors.
Department of Pathology, Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou 510089, China. wyan_sky@163.com
Yan
Y
Wang
Rui
R
Wang
Hui
H
Qiao
Jin-yun
JY
Li
Ting-sheng
TS
Peng
Yang
Y
Li
Meng
M
Zhang
Hui-zhen
HZ
Liang
Ju-shi
JS
Qiu
D001859:Bone Neoplasms / Q000378:metabolism* / Q000473:pathology; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D051793:Hypoxia-Inducible Factor 1; D020319:Oligodeoxyribonucleotides, Antisense; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D008156:Luciferases