hsa_circ_001160与PTBP1相互作用调控miR-195-5p影响血肿瘤屏障通透性的机制

The blood-tumor barrier (BTB) forms a major obstacle in brain tumor therapy by preventing the delivery of sufficient quantities of therapeutic drugs. Our previous work found that RNA binding protein PTBP1 and hsa_circ_001160 were up-regulated in the microvascular endothelial cells of human brain glioma. Down-regulation of PTBP1 or hsa_circ_001160 increased BTB permeability. Bioinformatics software predicted that there were PTBP1 and miR-195-5p binding sites in the hsa_circ_001160, and we briefly proved that hsa_circ_001160 in the regulation of BTB permeability might be associated with miR-195-5p. We also found that up-regulation of miR-195-5p or down-regulation of ETV1 increased BTB permeability, and meanwhile reduced the expression of ZO-1, occludin and claudin-5. Further, analysis of their promoter region showed the ETV1 binding sites, and there was a miR-195-5p binding site in the 3'-UTR of ETV1. These results suggested that miR-195-5p might increase BTB permeability by targeting ETV1, which could regulate the expression of ZO-1, occludin and claudin-5. Combined with our previous work, we inferred as follows: hsa_circ_001160 might interact with PTBP1, and inhibited the regulation of miR-195-5p targeting ETV1. Transcription factor ETV1 could positively regulate the expression of ZO-1, occludin and claudin-5 to regulate the BTB permeability. This project will firstly verify the binding and binding sites of PTBP1 in the hsa_circ_001160 to clarify the effects and mechanisms of hsa_circ_001160 in the regulation of BTB permeability with PTBP1. Further, we will verify the binding and binding sites of miR-195-5p in the hsa_circ_001160 and ETV1, and the effect of ETV1 on the transcriptional regulation of ZO-1, occludin and claudin-5 to prove the regulatory mechanism of BTB permeability. Finally, we will explore the best choice of increasing BTB permeability by administration with the inhibitor or activator separately or in combination, and combine with adriamycin to analyze the anti-tumor efficacy on glioma in nude mice. This project could not only clarify the molecular mechanisms of hsa_circ_001160 interacting with PTBP1 controlling miR-195-5p in the regulation of BTB permeability, but also reveal potential therapeutic targets for the treatment of brain glioma.

血肿瘤屏障(BTB)是影响化疗药物疗效的关键。前期发现人脑胶质瘤微血管内皮细胞中RNA结合蛋白PTBP1和hsa_circ_001160高表达，下调其表达均增加BTB通透性。通过前期工作推测高表达的hsa_circ_001160通过与PTBP1相互作用，减弱miR-195-5p对靶基因ETV1的调控，ETV1可调控紧密连接相关蛋白ZO-1、occludin和claudin-5的表达影响BTB通透性。项目先验证hsa_circ_001160与PTBP1的结合作用以及影响BTB通透性的效果与机制；进一步研究hsa_circ_001160与miR-195-5p，miR-195-5p与ETV1的靶向结合作用，阐明hsa_circ_001160通过miR-195-5p靶向ETV1调控紧密连接相关蛋白影响BTB通透性的机制；最后明确针对多个靶点的抑制剂或激动剂单独或联合应用增加BTB通透性的最佳方式。

脑胶质瘤是一种最常见的中枢神经系统恶性肿瘤，血肿瘤屏障(BTB)是影响化疗药物疗效的关键。首先验证hsa_circ_001160与PTBP1、hsa_circ_001160与miR-195-5p、miR-195-5p与ETV1的结合作用，并明确结合位点，进一步通过研究脑胶质瘤微血管内皮细胞中PTBP1、hsa_circ_001160、miR-195-5p和ETV1基因功能变化对紧密连接相关蛋白ZO-1、occludin和claudin-5的表达调节，明确上述基因或蛋白表达变化影响屏障通透性的分子机制。我们发现了下调hsa_circ_001160可通过与PTBP1相互作用，降低其与miR-195-5p的结合，增加miR-195-5p对靶基因ETV1的负性调控，下调的ETV1通过正性调控紧密连接相关蛋白ZO-1、occludin和claudin-5基因表达，增加血肿瘤屏障通透性，为增加脑胶质瘤的药物转运和提高疗效提供新途径。hsa_circ_001160、miR-195-5p与ETV1多个靶点的抑制剂或激动剂单独或联合应用增加血肿瘤屏障通透性的最佳方式，并联合应用化疗药物阿霉素，观察其对裸鼠脑胶质瘤的抑瘤效应。本项目的研究结果不仅能明确hsa_circ_001160通过与PTBP1相互作用影响血肿瘤屏障通透性的效果和机制，而且能为增加化疗药物转运进入肿瘤组织，切实提高脑胶质瘤的疗效提供新的靶点。