[Effect of silencing HIF-1alpha by RNA interference on expression of vascular endothelial growth factor in osteosarcoma cell line SaOS-2 under hypoxia].
OBJECTIVE
Hypoxia-inducible factor-1 alpha (HIF-1alpha) is a key regulator for hypoxia tolerance and angiogenesis of tumor. This study was to investigate the expression of HIF-1alpha and vascular endothelial growth factor (VEGF) in human osteosarcoma cell line SaOS-2 under hypoxia, to explore the effect of HIF-1alpha on hypoxia-activated angiogenesis regulation pathway in osteosarcoma.


METHODS
CoCl2 was used as chemical hypoxia-inducing reagent to mimic tumor hypoxic microenvironment. mRNA and protein levels of HIF-1alpha and VEGF at different hypoxic culture phases were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Small hairpin RNAs (shRNAs) eukaryotic expression vector targeting HIF-1alpha was constructed, and transfected into SaOS-2 cells. Western blot was used to detect gene silencing effect on HIF-1alpha. RT-PCR and enzyme-linked immunosorbent assay (ELISA) were used to observe the change of VEGF gene expression after HIF-1alpha gene silence.


RESULTS
Under hypoxia, mRNA level of HIF-1alpha kept stable, while its protein level increased obviouslyu both mRNA and protein levels of VEGF were up-regulated. The shRNAs plasmid targeting HIF-1alpha gene was constructed successfully, and down-regulated HIF-1alpha gene in SaOS-2 cells efficiently followed by VEGF gene down-regulation.


CONCLUSIONS
Hypoxia can increase protein level of HIF-1alpha in osteosarcoma. HIF-1alpha up-regulates the gene expression of VEGF via transcription activation which promotes angiogenesis in osteosarcoma under hypoxic microenvironment.
Wu
Qiang
Q
Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, P.R.China. wupower11@hotmail.com
Yang
Shu-Hua
SH
Wang
Rui-Ying
RY
Ye
Shu-Nan
SN
Xia
Tian
T
Ma
De-Zhang
DZ
D001859:Bone Neoplasms / Q000378:metabolism* / Q000473:pathology; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000235:genetics; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A