CRNDE靶向调控miR-186影响血肿瘤屏障通透性和肿瘤血管生长的分子机制

Colorectal neoplasia differentially expressed (CRNDE) gene, a long non-coding RNA gene, is the most upregulated lncRNA in gliomas. In our previous work, we found that the down-regulation of CRNDE inhibited the proliferation and migration of glioma cells, which suggested that CRNDE might act as an oncogene. There has been no report about the effects of CRNDE on the 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): inhibition of CRNDE, in brain glioma vascular endothelial cells, increased the BTB permeability. CRNDE might regulate the BTB permeability by interacting with miR-186 as a competitive endogenous RNA and inhibiting the miR-186 function. Overexpression of miR-186 in brain glioma vascular endothelial cells might reduce the the express of claudin-1 and occludin, and increase the BTB permeability. On the other hand, overexpression of miR-186 in brain glioma vascular endothelial cells might also decrease the express of essential autophagy gene beclin-1 and Atg7 directly or indirectly by regulating the express of transcription factor YY1 negatively, resulting in reduction of beclin-1 and Atg7 and inhibit of 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 CRNDE and miR-186. Then we will clarify the mechanisms of CRNDE on the regulation of BTB permeability, autophagy and angiogenesis via inhibiting miR-186 competitively. Further, we will verify the targeted binding and the binding sites between miR-186 and the target genes claudin-1 and occludin, and prove the regulation mechanism of miR-186 on BTB permeability. Moreover, the targeted binding and the binding sites between miR-186 and target genes YY1, beclin-1 and Atg7 will also be clarified, as well as the regulatory mechanism of miR-186 on autophagy of brain glioma vascular endothelial cells and angiogenesis. Finally, we will make a decision about the best choice between administration with CRNDE inhibitor and miR-186 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 regulation of BTB function and inhibition of tumor angiogenesis by the interaction of CRNDE and miR-186, but also provide a new pathway to strengthen the delivery of chemotherapeutic agents to neoplastic foci and enhance the therapeutic efficacy on brain gliomas.

通过前期工作提出假说：脑胶质瘤组织和肿瘤血管内皮细胞中高表达的结直肠肿瘤差别表达基因(CRNDE)通过与miR-186相互作用，抑制miR-186对靶基因的调节。MiR-186一方面负性调控胶质瘤微血管内皮细胞中紧密连接(TJ)相关蛋白claudin-1和occludin的表达，影响血肿瘤屏障(BTB)通透性；另一方面通过直接负性调控或通过转录因子YY1间接调控自噬相关蛋白beclin-1和Atg7的表达，抑制肿瘤血管生长。本项目拟先研究CRNDE抑制miR-186影响BTB功能，抑制肿瘤血管生长的机制；阐明miR-186调节BTB的机制；研究miR-186与YY1以及beclin-1和Atg7的靶向结合作用和位点，明确miR-186抑制肿瘤血管生长的机制；最后明确CRNDE 抑制剂、miR-186以及联合化疗药物增加BTB通透性、抑制肿瘤血管生长的效应。研究结果能为胶质瘤治疗提供新途径。

研究背景：我们的前期研究证实结直肠肿瘤差别表达基因(CRNDE)在脑胶质瘤组织和肿瘤血管内皮细胞中呈高表达状态，下调CRNDE的表达水平抑制了胶质瘤细胞的增殖和迁移。关于CRNDE是否能够调节血肿瘤屏障(BTB)的通透性、抑制肿瘤血管生长及其机制目前尚无报道。.主要研究内容：1）明确CRNDE和miR-186的靶向结合位点；2）阐明CRNDE抑制miR-186影响BTB开放，抑制肿瘤血管生长的机制；3）阐明miR-186调节BTB开放的机制；4）研究miR-186与YY1、beclin-1和Atg7的靶向结合作用和位点，明确miR-186抑制肿瘤血管生长的机制；5）明确CRNDE抑制剂、miR-186以及联合化疗药物增加BTB通透性、抑制肿瘤血管生长的作用。.重要结果：CRNDE通过与miR-186相互作用，1）抑制了miR-186对胶质瘤微血管内皮细胞上紧密连接蛋白的调节作用，进而影响BTB的通透性；2）通过直接负性调控或通过转录因子YY1间接调控自噬蛋白beclin-1和Atg7的表达，抑制肿瘤血管生长。.关键数据及科学意义：CRNDE抑制剂、miR-186以及联合化疗药物具有增强BTB通透性和抑制肿瘤血管生成的作用。本项目研究结果能够为脑胶质瘤的治疗提供新思路。