内皮-单核细胞激活多肽调控miR-429开放血肿瘤屏障的作用和机制

We have reported that endothelial monocyte-activating polypeptide-II (EMAP-II), selectively combined with α-ATP synthase on the tumor microvascular endothelial cells, could open the tight junction (TJ) through down-regulating the proteins expression of TJ associated proteins occludin and claduin-5, and then increase the permeability of blood-tumor barrier (BTB). In the following work, we found that EMAP-II could inhibit the mRNA expression levels of occludin and claudin-5. Meanwhile, we found that EMAP-II could increase the expression of miR-429 and inhibit the expression and the phosphorylation of p70S6K. But the mechanisms of EMAP-II down-regulating the expression of TJ associated proteins needs further research. Using molecular biologic and bioinformatics methods, we proposed the following working hypothesis building on a series of our previous research (see the details in the working foundation): EMAP-II might increase the permeability of BTB through up-regulating miR-429. Overexpression of miR-429 in brain glioma vascular endothelial cells might reduce the expression of occludin and claudin-5 directly in the post-transcriptional level. On the other hand, overexpression of miR-429 might decrease the expression of occludin and claudin-5 indirectly by regulating p70S6K negatively. To date, the mechanisms related to BTB permeability increase caused by EMAP-II, which maybe based on the up-regulation of miR-429 have not been reported yet.. In order to verify aboved-mentioned hypotheses, the first aim of this project is to study the effect of miR-429 during the process of EMAP-II regulating the expression of TJ associated proteins occludin and claduin-5 and opening the BTB. Secondly, to verify the targeted binding and the binding sites between miR-429 and its target genes occludin, claudin-5 and p70S6K, as well as the regulation of p70S6K on TJ associated proteins occludin and claudin-5 expression, to prove the mechanism of miR-429 on BTB permeability by directly targeted decreasing the expression of TJ associated proteins or indirectly decreasing the expression of TJ associated proteins via p70S6K. Finally, we will make a decision about the best choice between admistration with EMAP-II and miR-429 separately or in combination by analyzing the anti-tumor efficacy on glioma in nude rats with the associationof adriamycin. This project will not only illuminate the molecular mechanism of EMAP-II opening the BTB by regulating TJ, but also provide a theoretical basis for the clinical application of EMAP-II and new therapies for the integrated treatment of glioma as well.

我们报道了EMAP-II能通过下调紧密连接(TJ)相关蛋白表达开放血肿瘤屏障(BTB)。近期发现EMAP-II能上调miR-429的表达，antagomiR-429阻断了EMAP-II下调TJ相关蛋白表达、增加BTB通透性的作用；miR-429能显著抑制p70S6K的表达。建立工作假说如下：EMAP-II能通过上调miR-429，直接下调TJ相关蛋白表达，或通过p70S6K间接下调TJ相关蛋白表达，开放BTB。本项目拟首先研究miR-429在EMAP-II开放BTB中的作用；进一步验证miR-429与occludin、claudin-5和p70S6K存在靶向结合，以及p70S6K对TJ相关蛋白表达的调控，明确miR-429调控TJ结构和功能的机制；最后研究EMAP-II与miR-429单独或联合应用的效果。本项目不仅能明确EMAP-II开放BTB的机制，而且为脑胶质瘤的治疗提供新途径。

本项目通过系列研究发现并报道了EMAP-II通过miR-429增加血肿瘤屏障(BTB)的作用效果和机制。本项目研究结果显示：(1) EMAP-II能够通过开放紧密连接的细胞旁途径增加BTB通透性。(2) miR-429在脑胶质瘤微血管内皮细胞(GECs)低表达，并随病理级别的升高而降低；EMAP-II能够显著上调GECs中miR-429的表达。(3) miR-429过表达能够下调紧密连接相关蛋白ZO-1、occludin和claudin-5的mRNA和蛋白表达水平，并改变其分布，显著增加体外BTB模型通透性。(4) miR-429与ZO-1和occludin存在靶向结合作用，并明确了特异性结合位点，即证明了ZO-1和occludin分别是miR-429的靶基因；miR-429能够在转录后水平上抑制紧密连接相关蛋白ZO-1和occludin的表达。(5) EMAP-II能够显著下调p70S6K的表达水平和磷酸化水平；p70S6K过表达能够显著上调紧密连接相关蛋白ZO-1、occludin和claudin-5的mRNA和蛋白表达水平，并改变其分布，降低体外BTB模型通透性；p70S6K表达沉默则作用效过相反。(6) miR-429与p70S6K存在靶向结合作用，并明确了特异性结合位点，即证明了p70S6K是miR-429的靶基因；miR-429能够在转录后水平上抑制p70S6K达。(7) miR-429与p70S6K能够反向共同调节紧密连接相关蛋白ZO-1、occludin和claudin-5的表达，以及反向共同调节p70S6K-S6信号通路；证明了miR-429过表达能够通过下调p70S6K的表达和抑制p70S6K的活性，增加BTB通透性。(8) 裸鼠移植瘤实验发现，与EMAP-II和miR-429单独应用组相比，EMAP-II和miR-429联合应用是增加血肿瘤屏障通透性的最佳方式，能更有效地促进化疗药物阿霉素的转运，抑制胶质瘤的生长。本项目研究结果不仅明确了EMAP-II增加BTB通透性的分子机制，而且为脑胶质瘤的综合治疗提供了新靶点和新途径。