miRNA302-367通过多靶点抑制血管新生及其靶向肿瘤血管内皮在转移肿瘤治疗作用研究

The clinical application of VEGF antagonist in the treatment of metastasis tumor fails in long-term due to drug resistance. MiRNA represent a future therapeutic approach for the treatment of pathological neovascularization-related diseases because they target multiple pathways. Our preliminary data shows that miR302-367 are downregulated in tumor endothelial cells and endothelial specific overexpression of miR302-367 inhibits tumor angiogenesis. Gene expression profile by RNA sequencing shows that endothelial overexpression of miR302-367 downregulates cyclin D1 and rac1/cdc42, which control cell proliferation and cell migration respectively and play crucial roles in angiogenesis. Further study will confirm those genes are directly regulated by miR302-367. Endothelial specific viral vectors of these miR302-367 downstream target genes will be constructed to observe whether overexpression of these genes will reverse the phenotype of anti-angiogenesis and tumor growth inhibition by overexpression of miR302-367 in both in vitro cell cultures and in vivo mouse metastasis tumor models. An AAV virus vector under the sprouting endothelial specific apelin promoter will be constructed to achieve tumor sprouting endothelial specific overexpression of miR302-367. The long term therapeutic effect of sprouting endothelial specfic overexpression of miR302-367 in metastasis tumor will be tested and compared with VEGF antagonist. This study will help to elucidated that miR302-367 inhibits angiogenesis by targeting multi-pathway and providing new experimental evidence for the treatment of metastasis tumor.

临床VEGF拮抗剂抗肿瘤血管新生治疗因耐药致长期使用失效，故急需多靶点、靶向、高效抑制肿瘤血管新生新方法。通常miRNA调控多个基因发挥作用，推测miRNA将更有效抑制血管新生阻止肿瘤生长。我们前期结果发现肿瘤内皮细胞mir302-367表达下调，过表达mir302-367抑制肿瘤血管新生，表达谱测序结果发现内皮mir302-367过表达下调cyclin D1和Rac1/cdc42。我们进一步研究将证实mir302-367是否直接调控这些基因，过表达这些基因是否能够逆转mir302-367拮抗肿瘤血管新生阻碍肿瘤生长的作用，证实mir302-367可以调控多基因抑制血管新生。构建内皮出芽新生特异apelin启动子驱动的mir302-367腺相关病毒，观察肿瘤新生出芽内皮过表达特异mir302-367是否因调控多个靶基因较VEGF拮抗剂的长期效用更佳，为临床转移性肿瘤治疗提供新的思路。

恶性肿瘤是目前全世界的主要死亡原因之一，靶向肿瘤血管新生治疗是目前临床上肿瘤治疗的一大热点。临床上通过VEGF拮抗剂抗肿瘤血管新生治疗因耐药致使长期使用失效，因此急需多靶点、靶向、高效抑制肿瘤血管新生的新方法。.我们发现miR302-367在发育早期高表达随后逐渐降低，肿瘤内皮细胞中miR302-367表达下调。内皮过表达miR302-367后抑制小鼠胚胎期后脑及新生期视网膜血管新生，anti-mir302-367促进胚胎期血管新生。表达谱测序结果发现内皮miR302-367过表达后下调细胞骨架相关基因CDC42以及细胞周期相关基因CCND1。序列分析发现CDC42及CCND1 3’-UTR区域具有miR302-367互补序列，进一步机制研究证实miR302-367直接调控CDC42使其表达下调，并通过下游WASP信号调控F-actin形成以及通过KLF2-Grb2信号进一步调控PAK1/LIM-kinase/Cofilin信号对F-actin的聚合形成精确调控从而影响内皮细胞迁移以及出芽式血管形成。此外miR302-367通过直接调控CCND1使其表达下调，并导致使CCND1与CDK4蛋白结合下调引起RB磷酸化降低，从而抑制内皮细胞增殖及血管新生。小鼠皮下荷瘤实验证实miR302-367内皮过表达小鼠肿瘤生长显著抑制，肿瘤血管密度显著降低。.综上，本项目揭示了miR302-367通过多靶点抑制血管新生从而抑制肿瘤生长的作用机制，目前已构建了新生血管特异性启动子Apelin下的血管内皮细胞高亲嗜性RGDLRVS-AAV9表达载体，将进一步探索靶向内皮血管新生抑制转移瘤生长。探索转移性肿瘤防治新策略，为肿瘤治疗提供新思路与策略。