[Inhibitory effect of siRNA targeting HIF-1alpha on differentiation of peripheral blood endothelial progenitor cells]

Meng Jiang, Chang-Qian Wang, Bin-Yao Wang, Ding-Jiu Huang
Ai Zheng, Aizheng, Chinese Journal of Cancer 2005, 24 (11): 1293-300

BACKGROUND & OBJECTIVE: Transcription factor hypoxia inducible factor-1 alpha (HIF-1alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and hypoxia-induced pathophysiologic events. This study applied RNA interference targeting HIF-1alpha to human peripheral blood endothelial progenitor cells (EPCs) for therapeutic antineovascularization in vitro.

METHODS: Small interference RNA (siRNA) targeting HIF-1alpha was constructed and transfected into human EPCs. Transfection efficiency of siRNA-HIF-1alpha was measured by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry. The content of endothelial nitric oxide synthase (eNOS) was measured by ELISA. The morphology of EPCs/endothelial cells (ECs) cultured in normoxia or hypoxia was observed after siRNA-HIF-1alpha transfection.

RESULTS: SiRNA-HIF-1alpha was successfully constructed with the transfection efficiency of about 20%. After transfected for 6 h, HIF-1alpha mRNA was detected in EPCs cultured in normoxia or hypoxia with suppression; the expression of vascular endothelial growth factor (VEGF) was significantly attenuated (P < 0.05); the expression of HIF-1beta had no obvious change (P > 0.05). Stable expression of HIF-1alpha protein was induced by hypoxia, and was inhibited by siRNA-HIF-1alpha (P < 0.05). When cultured in 1% or 21% oxygen pressure for 3 days, CD31+ EPCs were inhibited more efficiently by siRNA-HIF-1alpha than by pEGFP (P < 0.05). The content of eNOS was significantly attenuated by siRNA-HIF-1alpha in time- and VEGF concentration-dependent manners (P < 0.01). EPCs differentiation and proliferation were induced by hypoxic culture, and suppressed by siRNA-HIF-1alpha.

CONCLUSIONS: The constitutive or hypoxia-induced expression of HIF-1alpha in EPCs is sufficient to affect the expression of downstream target genes. SiRNA targeting HIF-1alpha could inhibit the expression of the genes which promote neovascularization, and suppress the differentiation of EPC to EC.

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