miR-574-3p正反馈调控HIF/VEGF血管生成通路的分子机制研究

Cells can promote extracellular angiogenesis through an intracellular HIF / VEGF pathway, but the regulatory mechanism of this pathway has not been fully elucidated. Our previous work found that miR-574-3p induced up-regulation of vascular endothelial growth factor (VEGF) mRNA expression and down-regulation of mRNA and protein expression of cullin 2, an antagonist of hypoxia-inducible factor (HIF) ; Hypoxia increased intracellular HIF-1α protein content，upregulated miR-574-3p expression and downregulated cullin2 mRNA expression. In this project, we intend to prove that: 1) cullin 2 mRNA is a direct target of miR-574-3p; 2)miR-574-p inhibits the formation of E3 ubiquitin ligase complex through down-regulation of cullin 2, elevating intracellular HIF by inhibiting its degradation and activating the HIF/VEGF pathway to promote extracellular angiogenesis; 3) up-regulation of HIF by hypoxia promotes miR-574-3p expression which further increases activity of the HIF/VEGF pathway through an inhibition of cullin2 mRNA playing a role in positive feedback of hypoxia-induced extracellular angiogenesis. The completion of this project will elucidate the intracellular molecular pathway and regulatory mechanism of extracellular angiogenesis via miR-574-3p. We believe that further understanding of this pathway and regulatory mechanism would have significant practical and theoretical implications for angiogenesis-associated physiological or pathological processes such as in growth and development of blood vessel, wound healing, tumor development and cardio/cerebral vascular occlusion.

细胞通过HIF/VEGF通路促进周边血管生成，该通路调控机理尚未完全阐明。我们前期发现miR-574-3p上调VEGF表达，下调HIF对抗因子CUL2表达；缺氧升高细胞中HIF-1α蛋白含量、上调miR-574-3p表达、下调CUL2表达。本项目中我们拟阐明：1）CUL2是miR-574-3p的靶基因；2）miR-574-3p通过抑制CUL2抑制细胞内E3-泛素连接酶复合物的形成，解除后者介导的HIF-1α降解，激活HIF/VEGF通路，促进细胞外血管生成；3）HIF-1α上调miR-574-3p的表达，后者通过抑制CUL2进一步加强HIF/VEGF通路的活性，对缺氧诱发的细胞外血管生成形成正反馈调控作用。本项目的完成将阐明细胞内miR-574-3p促进细胞周围血管生成的分子通路及调控机制，对血管生长和发育、组织创伤愈合、心脑血管阻塞后血管再生、肿瘤组织血管生成等具有普遍的理论和实践意义。

细胞通过HIF/VEGF通路以旁分泌的方式促进细胞周围血管生成，该通路调控机理尚未完全阐明。MicroRNA是新近发现的一类重要的转录后调控因子，广泛参与了机体的生理和病理过程，包括血管生成。在本项目研究中，我们阐明了胃癌细胞中miR-574-3p正反馈调控HIF/VEGF血管生成通路的分子机制。我们的研究结果表明，缺氧条件下，胃癌细胞HIF-1α蛋白的水平升高，HIF-1α蛋白能够直接启动miR-574基因的转录，提高细胞中miR-574-3p的水平。MiR-574-3p通过直接抑制胃癌细胞中CUL2蛋白的表达，并由此抑制细胞中HIF-1α蛋白的泛素化降解过程，从而稳定细胞中HIF-1α蛋白水平，然后通过HIF/VEGF通路促进血管的生成。本项目研究不仅丰富了血管生成调控机制的理论，也为通过阻断血管生成从而治疗肿瘤提供了潜在的靶点。