[Effect of silencing HIF-1alpha by RNA interference on expression of vascular endothelial growth factor in osteosarcoma cell line SaOS-2 under hypoxia]

Qiang Wu, Shu-Hua Yang, Rui-Ying Wang, Shu-Nan Ye, Tian Xia, De-Zhang Ma
Ai Zheng, Aizheng, Chinese Journal of Cancer 2005, 24 (5): 531-5

BACKGROUND & 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.

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