MIAT与miR-140-3p调控ZAK基因表达调节血肿瘤屏障通透性的机制

Myocardial infarction associated transcript (MIAT) gene, a long non-coding RNA gene, is considered to have contacted with the certain biological characteristics of endothelium and / or epithelium. In our previous work, MIAT expression in human cerebral microvascular endothelial cell/D3 (hCMEC/D3) of blood-tumor barrier (BTB) was significantly increased, and the permeability in BTB was significantly higher than the permeability in blood-brain barrier (BBB). Further silence the expression of MIAT in hCMEC/D3 of BTB, the permeability in BTB significant decreased. A decrease in miR-140-3p expression was detected by gene chip and real-time PCR. Similary, miR-140-3p is considered to join in regulation of BTB permeability in our previous study . There has been no report about the effect of MIAT and miR-140-3p on the regulation of BTB permeability, as well as the related potential mechanisms. Using molecular biologic and bioinformatics methods, we proposed the following working hypothesis on the basis of the preliminary work: Effect of MIAT and miR-140-3p on the expression of ZAK regulate blood-brain barrier permeability. MIAT and miR-140-3p bind to ZAK in the 5' upstream promoter region and 3'-UTR respectively and regulate ZAK expression, and MIAT as a competing endogenous RNA (ceRNA) further inhibit the function of miR-140-3p，which results to an increase in ZAK expression. ZAK regulates the BTB permeability through competitive inhibition of ZONAB effect and negative transcription for tight junction (TJ) associated proteins.. On the basis of our previous work, we will firstly demonstrate whether MIAT as a ceRNA inhibits the function of miR-140-3p, which results to an increase in ZAK expression in this project. Then, we will demonstrate whether there is a binding of MIAT and the RNA-binding domain (RBD) of EWSR1 protein, which results to change the EWSR1 conformation and activate EWSR1. Activated EWSR1 protein in the nuclear as a transcription factor could play a role in the positive regulation of ZAK expression via EWS-Transcriptional Activation-Domain (EAD). Further，this project will verify direct interaction and key binding sites between the seed sequence of miR-140-3p and ZAK mRNA-3'UTR target sites using luciferase reporter assay. We will research the transcriptional regulation mechanism of miR-140-3p on ZAK and clarify its functions and its impact on the structure and function of BTB. Moreover we will demonstrate whether ZAK could make a simultaneous distribution in ZONAB from TJ to nucleus via competitive inhibition, and ZONAB in nucleus as a negative transcription factors could regulate the expression of TJ associated proteins claudin5, occludin and ZO-1. We will clarify the mechanisms of ZAK as a negative transcription factors regulating the expression of TJ associated proteins claudin5, occludin and ZO-1 via leucine-zipper. Finally, we will make a decision about the best choice between administration with overexpression of MIAT and the downexpression of miR-140-3p separately or in combination by analyzing the anti-tumor efficacy on glioma in nude rats with the association of adriamycin. This project could not only clarify the molecular mechanisms related to the effect of MIAT and miR-140-3p on the expression of ZAK regulating blood-brain barrier permeability, but also provide a new pathway to strengthen the delivery of chemotherapeutic agents to brain gliomas and enhance the therapeutic efficacy on brain gliomas.

通过前期工作提出假说：MIAT与miR-140-3p分别和ZAK的5’端上游启动子区和3’-UTR相互作用调控其表达，MIAT作为ceRNA进一步抑制miR-140-3p作用使ZAK表达升高，ZAK通过竞争性抑制ZONAB作用和负性转录因子作用调控BTB通透性。项目首先研究MIAT作为ceRNA竞争性抑制miR-140-3p作用，通过激活EWSR1增加ZAK启动子转录活性的机制，研究miR-140-3p对ZAK转录后负性调控机制；进一步研究ZAK通过竞争性抑制ZONAB作用和负性转录因子作用调控TJ相关蛋白表达机制，最后研究过表达MIAT和表达沉默miR-140-3p单独或联合应用增加BTB通透性最佳方式，靶向感染脑胶质瘤BMEC，联合应用化疗药物，观察其对裸鼠抑瘤效应。本项目不仅能够揭示ncRNA调控ZAK表达调节BTB通透性机制，而且能够为增加化疗药物转运、提高脑胶质瘤疗效提供新途径。

脑胶质瘤是中枢神经系统一种常见的恶性肿瘤，其生长迅速，术后复发率 高，术后化疗是延长脑胶质瘤患者生存期的一种有效手段。血肿瘤屏障(blood-tumor barrier, BTB) 主要由脑微血管内皮细胞、基膜和肿瘤细胞构成，由于 BTB 的存在，极大限制了化疗药物进入肿瘤组织，很大程度上影响了化疗效果。有研究显示在脑组织中化疗药物的浓度提高1倍，那么化疗效果将提高10倍，因此国内外学者一直致力于探寻能够增加 BTB 的通透性，促进化疗药物进入肿瘤组织的有效方法。.在本研究中，我们首次证实了MIAT在GECs中高表达，高表达的MIAT增加BTB的通透性。MiR-140-3p在GECs中低表达，低表达的miR-140-3p对ZAK起转录后负性调控作用进而使ZAK的表达增加。高表达的MIAT发挥ceRNA作用进一步抑制的miR-140-3p功能，从而加强上述效应，使BTB的通透性增加。高表达的ZAK磷酸化NFκB使其转录抑制活性增强，通过对紧密连接相关蛋白ZO-1、occludin和claudin-5起负性转录因子作用，使TJ相关蛋白表达水平降低，进而增加BTB的通透性，表达沉默miR-140-3p和过表达MIAT联合应用使阿霉素诱导胶质瘤细胞凋亡率明显升高。本研究的主要目的是明确MIAT通过对miR-140-3p发挥ceRNA作用调控血肿瘤屏障通透性的机制，为增加BTB通透性进而提高脑胶质瘤的化疗效果提供新思路和新靶点。