miR-34家族调节血肿瘤屏障通透性和肿瘤血管生长的作用与分子机制

Both foreign reports and our previous work proved the expression levels of miR-34 family are low in gliomas and tumor vascular endothelial cells, which acts as tumor suppressor genes to regulate the biological behavior of glioma. There has been no report about the effects of miR-34 family membranes on regulation of blood-tumor barrier (BTB) permeability and inhibition of tumor angiogenesis, as well as the related potential mechanisms. 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): overexpression of miR-34a and miR-34c, the membranes of miR-34 family, in brain glioma vascular endothelial cells, might regulate the express of transcription factor MAZ negatively, resulting in reduction of the tight junction associated proteins claudin-5, occludin, ZO-1 and increase of BTB permeability. On the other hand, miR-34a and miR-34c might also change the express and distribution of tight junction associated proteins and cytoskeletal proteins by regulating the express of PKCε negatively, causing further increase of the BTB permeability. Meanwhile, overexpression of miR-34a and miR-34c in brain glioma vascular endothelial cells might also induce autophagy to inhibit tumor angiogenesis..On the basis of our previous work, this project will firstly verify the targeted binding and the binding sites between miR-34a and miR-34c and their target genes MAZ and PKCε. Then we will clarify the regulatory mechanisms of miR-34a and miR-34c on MAZ gene express, and prove the transcriptional regulatory mechanisms of MAZ on tight junction associated proteins as well as the effects on BTB function. Moreover, the regulatory mechanisms contributing to PKCε expression, BTB function, autophagy and tumor angiogenesis by miR-34a and miR-34c will be investigated. Finally, we will make a decision about the best choice between administration with miR-34a and miR-34c separately or in combination by analyzing the anti-tumor efficacy on glioma in nude rats with the association of carboplatin. This project could not only clarify the molecular mechanisms related to the regulation of BTB function and inhibition of tumor angiogenesis by miR-34a and miR-34c, but also provide a new pathway to strengthen the delivery of chemotherapeutic agents to neoplastic foci and enhance the therapeutic efficacy on brain gliomas.

国外报道和我们前期工作证明miR-34家族在胶质瘤组织和肿瘤血管内皮细胞中低表达，发挥抑癌基因的作用。miR-34家族调节血肿瘤屏障(BTB)通透性和抑制肿瘤血管生长的效果及机制未见报道。通过系列前期研究提出了工作假说：在脑胶质瘤微血管内皮细胞中过表达miR-34家族成员miR-34a和miR-34c，可能分别通过负性调节转录因子MAZ和PKCε的表达增加BTB通透性；同时激活自噬反应抑制肿瘤血管生长。本项目拟先验证miR-34a和miR-34c分别与MAZ和PKCε的结合，明确结合位点；研究miR-34a和miR-34c通过调控MAZ基因表达影响BTB功能的机制；进一步研究miR-34a和miR-34c通过调控PKCε基因表达影响BTB功能、自噬反应和肿瘤血管生长的机制；最后明确miR-34a或miR-34c单独或联合应用的效果。研究结果能为增加化疗药物转运，提高脑胶质瘤疗效提供新途径。

本项目通过系列研究在国内外率先发现并报道了miR-34家族调节血肿瘤屏障通透性和肿瘤血管生长的作用与分子机制。新发现如下：(1)在脑胶质瘤微血管内皮细胞中miR-34家族三个成员miR-34a、miR-34b、miR-34c的均呈低表达；分别在血管内皮细胞中过表达miR-34a、miR-34b和miR-34c，只有miR-34a和miR-34c的过表达能显著增加血肿瘤屏障通透性，miR-34b 对血肿瘤屏障通透性无明显作用。(2) miR-34a和miR-34c分别与MAZ和PKCε存在靶向结合作用，并明确了特异性结合位点，即证明了MAZ和PKCε分别是miR-34a和miR-34c 的靶基因。(3) miR-34a和miR-34c分别通过调控MAZ和PKCε的基因表达，影响紧密连接相关蛋白ZO-1、Occludin和Claudin-5的转录和表达，以及在细胞内的分布，通过细胞旁途径调控血肿瘤屏障通透性。(4) MAZ作为转录因子分别与ZO-1、Occludin和Claudin-5的启动子区的特异序列结合，影响ZO-1、Occludin和Claudin-5的转录。(5)下调MAZ的表达，显著增加了血肿瘤屏障通透性。(6)miR-34a和miR-34c分别通过激活PKCε，改变PKCε活性，以及增加PKCε的表达调节血肿瘤屏障通透性。(7)过表达miR-34a和miR-34c抑制了胶质瘤微血管内皮细胞的增殖、迁移和管形成能力，其机制与显著增加LC3 II/I和beclin 1的表达，以及降低p62的表达相关，即证明了过表达miR-34a通过激活胶质瘤微血管内皮细胞的自噬反应抑制了血管生长。(8)通过裸鼠移植瘤实验发现，与单独应用Ad-miR-34a、Ad-miR-34c、Ad-siRNA-MAZ组相比较，Ad-miR-34a和Ad-miR-34c联合应用组增加血肿瘤屏障通透性和抑制肿瘤血管生长的效果最佳。上述研究结果不仅为miRNA调控脑微血管内皮细胞的功能提供了新的理论依据，也为探索促进大分子药物选择性通过血肿瘤屏障，进入脑胶质瘤组织，切实提高脑胶质瘤的化疗疗效提供了新靶点和新策略。