Study of gene therapy with VEGF RNA interference against oral cancer

双链RNA(DsRNA)在RNA干扰(RNAi)中起着重要作用，在RNAi过程中，dsRNA的片段最初被DICER切割成更短的片段(21-23nt)，称为小干扰RNA(SiRNA)。然后，这些siRNA专门针对同源的mRNA分子，使它们被细胞核糖核酸酶降解。RNAi下调哺乳动物细胞内源性基因的表达。血管内皮生长因子是血管生成的关键分子，也是血管生成的关键分子。肿瘤的生长是一个依赖血管生成的过程，以抑制血管生成为目的的治疗策略在理论上是有吸引力的。为了研究针对血管内皮生长因子的siRNA用于特异性下调血管内皮生长因子信使核糖核酸，从而抑制血管生成的可行性，我们利用以血管内皮生长因子为靶点的小干扰RNA系统的DNA载体进行了实验。几乎完全抑制三种不同亚型(VEGF120、VEGF164和VEGF188)在小鼠鳞状细胞癌NRS-1细胞中的表达和蛋白的分泌。阳离子明胶微球释放的siVEGF体内抑制肿瘤生长。血管显着减少伴随着siVEGF转基因肿瘤的抑制。荧光显微镜观察显示，注射后10d，肿瘤周围仍可见siVEGF与阳离子明胶微球的复合体存在，而游离siVEGF已消失。此外，siVEGF基因治疗增加了周细胞覆盖血管的比例，并诱导周细胞表达血管生成素-1。这些数据表明，含有siVEGF的阳离子明胶微球可用于在NRS-1鳞癌异种移植模型中使肿瘤血管正常化并抑制肿瘤生长。

Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down-regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive.In order to investigate the feasibility of using the siRNA for VEGF in the specific knock-down of VEGF mRNA, thereby inhibiting angiogenesis, we have done experiments with a DNA vector based on a small interfering RNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. Furthermore, siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in an NRS-1 squamous cell carcinoma xenograft model.