干扰RNA沉默HIF-1α在缺氧状态下对骨肉瘤细胞VEGF表达的影响

Effect of Silencing HIF-1α by RNA Interference on Expression of Vascular Endothelial Growth Factor in Osteosarcoma Cell Line SaOS-2 under Hypoxia

背景与目的:研究表明H IF-1α是肿瘤适应低氧微环境、诱导血管新生的一个主要调控因子。本研究通过观察体外低氧培养条件下骨肉瘤细胞系SaOS-2中H IF-1α和VEGF的表达,探讨H IF-1α在骨肉瘤缺氧激活血管新生调控途径中的作用。方法:CoCl2化学缺氧法模拟肿瘤缺氧环境。半定量RT-PCR和免疫组化法分别检测不同缺氧时相中H IF-1α和VEGF在m RNA和蛋白水平的表达。构建针对H IF-1α的短发夹状小干扰RNA真核表达载体并转染SaOS-2细胞。免疫印迹沉淀观察转染后H IF-1α的基因沉默效果,RT-PCR和ELISA双抗体夹心法检测H IF-1α短基因沉默后SaOS-2细胞中VEGF的变化。结果:低氧条件下,SaOS-2细胞H IF-1αm RNA水平稳定,蛋白表达显著升高;而VEGF m RNA和蛋白的表达均显著升高。构建的H IF-1α短发夹状小干扰RNA表达载体转染SaOS-2细胞后能够显著下调H IF-1α基因的表达,同时VEGF基因的表达也受到明显抑制。结论:缺氧促使SaOS-2细胞H IF-1α在蛋白水平表达升高,H IF-1α通过转录激活VEGF的机制促进骨肉瘤缺氧状态下的血管新生。

BACKGROUND & OBJECTIVE: Hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulator for hypoxia tolerance and angiogenesis of tumor. This study was to investigate the expression of HIF-1α and vascular endothelial growth factor (VEGF) in human osteosarcoma cell line SaOS-2 under hypoxia, to explore the effect of HIF-1α 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-1α 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-1α was constructed, and transfected into SaOS-2 cells. Western blot was used to detect gene silencing effect on HIF-1α. RT-PCR and enzyme-linked immunosorbent assay (ELISA) were used to observe the change of VEGF gene expression after HIF-1α gene silence. RESULTS: Under hypoxia, mRNA level of HIF-1α kept stable, while its protein level increased obviously; both mRNA and protein levels of VEGF were up-regulated. The shRNAs plasmid targeting HIF-1α gene was constructed successfully, and down-regulated HIF-1α gene in SaOS-2 cells efficiently followed by VEGF gene down-regulation. CONCLUSIONS: Hypoxia can increase protein level of HIF-1α in osteosarcoma. HIF-1α up-regulates the gene expression of VEGF via transcription activation which promotes angiogenesis in osteosarcoma under hypoxic microenvironment.