Antitumor effect utilizing antiangiogenic activity by RNAi as new gene knock-down method

利用 RNAi 的抗血管生成活性作为新的基因敲除方法的抗肿瘤作用

• 批准号: 15390420
• 负责人: FUKAI Ichiro
• 金额: $ 9.28万
• 依托单位: NAGOYA CITY UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390420/
• 关键词: VEGF; RNAi; Antiangiogenesis; Antitumor effect; 血管新生; VEGFレセプター; in vivo形質導入

It has been known that tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of antiangiogenic factors should be a viable approach for cancer gene therapy. Similarly, inhibition of angiogenic factor has an impact to suppress the tumor growth. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and many studies have shown that VEGF is upregulated in many human tumors. We have planned to inhibit vascular VEGF receptors using RNA interference (RNAi). RNAi is. a powerful tool to silence gene expression post-transcriptionally. The strategy to regulate the tumor angiogenesis with RNAi technique targeted VEGF in tumor cell. We investigated the silencing effect of small. interfering RNA (siRNA) duplexes targeting the gene VEGF-receptor (VEGFR) in vascular endothelial cell to inhibit its activity of angiogenesis and evaluated its effect against to the tumor progression in vivo model. We optimized the RNAi target sequence.of VEGFR-1. Transfection of VEGFR-1 siRNA to vascular endothelial cell specialy reduced VEGFR-1 mRNA level and inhibit the proliferation of transfected cell. The vector that express short hairpin RNAs (shRNA) working as the siRNA duplexes to VEGFR-1 gene was constructed. These vectors specially reduced VEGFR-1 expression. Further, the RNAi target sequence to VEGFR-2 was optimized and the shRNA targeted to VEGFR-2 expression vector was constructed. We investigated this powerful utility in in vivo model. The experiment showed that VEGFR-2-shRNA inhibited tumor progression. So we plan to evaluate the inhibitory effect in in vivo model of VEGFR-1-shRNA and mix of two kind of shRNA.

众所周知，肿瘤需要持续的血管生成来支持其生长。通过产生抗血管生成因子来抑制血管生成应该是癌症基因治疗的一个可行的方法。同样，抑制血管生成因子也有抑制肿瘤生长的作用。血管内皮生长因子(VEGF)是肿瘤血管生成的关键调节因子，许多研究表明，在许多人类肿瘤中，VEGF表达上调。我们已经计划使用RNA干扰(RNAi)来抑制血管内皮细胞生长因子受体。RNAi是。转录后抑制基因表达的强大工具。靶向肿瘤细胞内血管内皮生长因子的RNAi技术调控肿瘤血管生成的策略。我们考察了Small的消声效果。针对血管内皮细胞中血管内皮生长因子受体(VEGFR)基因的干扰RNA(SiRNA)抑制其血管生成活性，并在体内模型中评价其抗肿瘤进展的作用。我们对VEGFR-1的RNAi靶序列进行了优化。将VEGFR-1siRNA导入血管内皮细胞，可显著降低VEGFR-1mRNA水平，抑制细胞增殖。构建了VEGFR-1基因的siRNA双链短发夹状RNA表达载体。这些载体特别降低了VEGFR-1的表达。进一步优化了针对VEGFR-2的RNAi靶序列，构建了针对VEGFR-2的shRNA表达载体。我们在活体模型中研究了这种强大的效用。实验表明，VEGFR-2-shRNA抑制了肿瘤的发展。因此，我们计划在体内模型中评价VEGFR-1-shRNA以及两种shRNA的混合物的抑制作用。