Lnc00462717与PTBP1相互作用调控miR-186开放血脑肿瘤屏障的作用与机制研究

How to selectively open the blood-brain tumor barrier (BTB) is an emerging strategy for the efficient treatment of brain tumor. Caveolin-1 plays an important role on regulating the permeability of BBB. FoxO1 is one of transcription factors, which can combine with DNA sequences 5’-TTGTTTAC-3′of promoters, and then regulate the transcription of down-stream target genes. The previous study demonstrated that FoxO1 might regulate the transcription of caveolin-1. FoxO1 is an important effector for PI3K/PKB signal pathway and can on-off many genes transcription by phosphorylation and dephosphorylation. PTEN is an important negative regulator of the PI3K/PKB pathway. Overexpression of PTEN can inhibit PI3K/PKB activity and the phosphorylation of FoxO1, which enhances the transcriptional activity. PTEN interacted with caveolin-1, which could be up-regulated by overexpression of caveolin-1. The results suggest that FoxO1 induced the upregulation of caveolin-1, while the phosphorylation of FoxO1 might be significantly inhibited by PTEN. SIRT1 promotes the deacetylation of Lysine residues in DNA binding sequence of FoxO1 and induces the location from the nuclear to the cytoplasm. Moreover, SIRT1 activation or overexpression can induce the expression of PTEN protein. Therefore, we speculate that“Caveolin-1 regulating-circuit”is formed among SIRT1, FoxO1, caveolin-1, PTEN, PI3K and PKB. And SIRT1, FoxO1 and PTEN are important and regulative proteins in “Caveolin-1 regulating-circuit”. We demonstrated that lnc00462717 interacts with PTBP1 and could significantly increase the permeability of BTB. The PTBP1 binding sites were found in the 3’UTR of FoxO1, PTEN and SIRT1 promoters targeted to miR-186. Therefore, we hypothesize that lnc00462717 interacts with PTBP1 and regulates the “Caveolin-1-regulating-circuit”by targeting to miR-186 and then selectively opens the permeability of BTB. The first aim of this study is to investigate the effect of miR-186 on the “Caveolin-1-regulating-circuit”; further study will focus on the role of lnc00462717 in miR-186-mediated regulating on “Caveolin-1-regulating-circuit”. Finally, we study that the effect of lnc00462717 on the transportation of antitumor drug and treatment of glioma in cavelin-1 (+/-) mice. This is the first study to reveal the mechanism of lnc00462717 in selectively regulating the BTB, and providing the theoretical and experimental evidences to fully understand the related mechanisms, and create a new target for drug development in brain glioma and other diseases of the central nervous system.

如何选择性开放血脑肿瘤屏障(BTB)是研究有效治疗脑肿瘤的焦点。Cav-1在BTB通透性调节中发挥重要作用。课题组前期证实：lnc00462717与PTBP1存在相互作用；"Cav-1调节环路”中关键调控蛋白的3’UTR区均存在PTBP1结合位点，且均与miR-186相结合;lnc00462717显著增加BTB通透性。我们推测lnc00462717与PTBP1相互作用调控miR-186开放BTB。本项目首先研究miR-186对“Cav-1调节环路”的影响；进一步研究lnc00462717与PTBP1互作在miR-186调控“Cav-1调节环路”中的作用；最后研究lnc00462717联合抗肿瘤药物治疗cav-1(-/-)和cav-1(+/+)脑胶质瘤小鼠的效果及机制。本研究首次揭示lnc00462717与PTBP1相互作用开放BTB的作用机制，为脑胶质瘤等中枢神经系统疾病的治疗开创新靶点。

胶质瘤是一种常见的中枢神经系统肿瘤，具有致死率高、治愈率低、复发率高、预后效果差等特点。目前临床治疗以手术为主，放疗、化疗以及分子靶向治疗为辅。但由于血脑肿瘤屏障 (Blood-Brain Tumor Barrier, BBB/BTB) 的存在大大降低了化疗药物药效，因此改善化疗药物的BTB传递效率成为胶质瘤治疗中亟需解决的关键问题。本研究使用人脑微血管内皮细胞 hCMEC/D3与人脑恶性胶质母细胞瘤细胞U87-MG共培养，构建体外BTB模型，利用超快速液相色谱-质谱系统检测Lnc00462717和agomiR-186-5p对大鼠脑胶质瘤以及瘤周组织中阿霉素累积量的影响。研究发现 GECs中表达上调的Lnc00462717、PTBP1和miR-186-5p均可通过调控靶定基因特定序列参与体外血脑肿瘤屏障通透性的调节。进一步证实 Lnc00462717通过与PTBP1直接结合调控体外BTB通透性； PTBP1和miR-186-5p均与靶定基因的3’ UTR 结合；Lnc00462717/PTBP1一方面减低内源性miR-186-5p表达，另一方面解除miR-186-5p对靶基因的负性调控作用，调控体外BTB通透性；最后通过裸鼠原位移植瘤实验证实：Lnc00462717/miR-186-5p调控抗肿瘤药Dox在胶质瘤组织中的累积量。因此，Lnc00462717和miR-186-5p可作为开放BTB和治疗神经胶质瘤药物的潜在靶点，本研究为选择性开放BTB通透性及胶质瘤治疗的药物研究提供新思路。

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