RMP-7通过Caveolin-1调控血肿瘤屏障通透性的分子机制

RMP-7 selectively opens blood-tumor barrier and shows promising efficacy in the preclinical application of glioma, but the mechanism is unclear. Our previous work proved that the mRNA level of caveolin-1 raised after treatment with RMP-7; meanwhile NF-κB and caveolin-1 were found to colocalize in the cytoplasm of rat brain microvascular endothelial cells. Analyzed by bioinformatics, we found that caveolin-1 transcription regulation sequence (Genebank NM_001753.3) contains four NF-κB binding sites, which were located in -55~-46、+486~+495、+1873~+1884 and +1966~+1975 base sites. Electrophoretic Mobility Shift Assay demonstrated that -55~-46 base site owned nuclear protein binding ability. On this basis, we cloned a part of caveolin-1 gene transcription regulatory sequences. Luciferase Assay confirmed preliminarily that caveolin-1 gene promoter should be located in -250 bp~ -40bp, and has a positive regulation element in -40bp~+600bp. The results of Co-Immunoprecipitation showed that NF-κB(p65) and caveolin-1 could interact, but this interaction was weakened in cytoplasm of endothelial cells after treated of RMP-7. Based on synthetically analysis of domestic and foreign researches and our previous studies, we assumed that NF-κB interacted with caveolin-1 through the caveolin binding motif, and the dissociation between them was induced by RMP-7 through Ca2+ pathway. Then NF-κB translocated into the nucleus to regulate the transcription of caveolin-1 gene, leading to an increase of caveolin-1 protein expression. Increased caveolin-1 inversed into cell membrane to promote transcytosis and to increase BTB permeability. After retrieval in domestic and abroad, the following working hypothesis had not been reported yet. NF-κB could interact with caveolin-1 through the caveolin binding motif. The dissociation between NF-κB and caveolin-1 was induced by RMP-7 through increasing Ca2+ levels and promoting NF-κB to translocate into nucleus to regulate caveolin-1 transcription and increase caveolin-1 expression.. This project firstly intended to explore the effects of RMP-7 on the activation of NF-kB signaling and the regulation by NF-κB about the BTB structure, function and caveolin-1 mRNA and protein expression to make clear the role of NF-κB in the selective opening of BTB by RMP-7. Based on this, the project intended to study whether NF-κB interacted with caveolin-1 through the caveolin binding motif and investigated the effects of signaling molecule Ca2+ on the combination between NF-κB and caveolin-1 to make clear the mechanism that how RMP-7 regulate the interaction between NF-κB and caveolin-1. Finally, the project intended to investigate the effects and mechanism of NF-κB on caveolin-1 transcriptional regulation. The project will not only identify the molecular mechanisms how RMP-7 regulate caveolae protein caveolin-1, but also provide new targets for the treatment of central nervous system diseases such as glioma.

选择性开放血肿瘤屏障的RMP-7在脑胶质瘤的临床前期应用中取得了良好的效果，但机制不清。前期研究发现，RMP-7作用下NF-κB与caveolin-1在大鼠脑微血管内皮细胞的胞浆中存在共定位，NF-κB蛋白的氨基端末端间存在caveolin-结合基序。为明确NF-κB对caveolin-1的调控机制，本项目首先研究RMP-7对NF-κB的调节，以及NF-κB对BTB结构和功能、caveolin-1表达的影响；进一步研究NF-κB是否通过caveolin-结合基序与caveolin-1相互作用，探讨Ca2+对NF-κB与caveolin-1结合的影响，明确RMP-7调节NF-κB与caveolin-1相互作用的机制；最后分析NF-κB对caveolin-1的转录调控作用和机制。本项目不仅能明确RMP-7调节质膜微囊结构蛋白caveolin-1的分子机制，而且能够为脑胶质瘤的治疗提供新靶点。

本项目通过系列研究阐释了缓激肽类似物RMP-7通过跨细胞途径调控血肿瘤屏障通透性的分子机制。取得研究成果如下：(1) RMP-7上调了血肿瘤屏障内皮细胞中NF-κB的活性和表达，并改变了细胞内的分布。(2) RMP-7诱导的NF-κB活性和表达增加与质膜微囊结构蛋白caveolin-1的mRNA和蛋白表达上调、吞饮小泡数量增加以及血肿瘤屏障通透性的增加呈正相关关系，RMP-7可通过NF-κB途径增加吞饮小泡的数量，即通过跨细胞途径选择性开放血肿瘤屏障。 (3) NF-κB过表达后能够显著增加体外BTB模型的通透性、caveolin-1的表达，并且NF-κB与caveolin-1的结合能力显著增强。(4) NF-κB能够通过caveolin-结合基序与caveolin-1结合形成复合物NF-κB-caveolin-1，此复合物是NF-κB介导的跨细胞途径开放血肿瘤屏障的基础。(5) RMP-7通过促进细胞内Ca2+释放，诱导NF-κB-caveolin-1复合物解离，促进NF-κB转位入核，caveolin-1进入细胞膜。 (6) NF-κB作为转录因子能够与caveolin-1的启动区的转录调控序列结合，影响caveolin-1的基因转录。(7) 通过裸鼠移植瘤实验发现，与NF-κB重组腺病毒载体、化疗药物(卡铂)单独应用组相比较，二者联合应用的抑瘤效果更佳。证明了NF-κB能够增加BTB通透性，促进化疗药物转运，抑制脑胶质瘤的生长。本研究结果不仅明确了RMP-7调节质膜微囊结构蛋白caveolin-1的分子机制，而且为脑胶质瘤等中枢神经系统疾病的治疗提供了新靶点。