circRNA_0011721-miR-519a-STAT3正反馈回路调控自噬促进胶质母细胞瘤化疗耐药的机制研究

Autophagy in glioblastoma (GBM) is closely related to temozolomide (TMZ) chemoresistance. We have demonstrated that miR-519a /STAT3 pathway plays an important role in the regulation of autophagy and chemoresistance of TMZ in GBM, but the regulatory mechanism of this pathway is not yet clear. We previously identified a circular RNA，named circ_0011721,is related to miR-519a expression and chemoresistance of TMZ by circRNA microarray and StarBase database. Further research showed that circ_0011721 colocalized with miR-519a, and circ_0011721 inhibited autophagy formation. In addition, bioinformatics analysis revealed that STAT3 could bind to the promoter region of MTF1,which is host gene of circ_0011721. Therefore, we proposed a hypothesis:circ_0011721/miR-519a/STAT3 form a positive feedback loop to inhibit the formation of autophagy in GBM cells, thereby promoting drug resistance. In this project, we will analyze the effect of circ_0011721 on TMZ sensitivity and autophagy of GBM cells and the molecular mechanisms of circ_0011721 regulation of miR-519a in vitro and in vivo through FISH、luciferase assay、ChIP and RIP assays. We can further understand the molecular mechanisms of circ_0011721 involved in GBM chemoresistance, and provide a new candidate target for the comprehensive treatment of GBM.

胶质母细胞瘤（GBM）化疗过程中替莫唑胺（TMZ）耐药与自噬密切相关。申请人前期已证实miR-519a通过抑制STAT3促进自噬来增强GBM化疗敏感性，但miR-519a上游调控机制不清。本项目前期筛选到环状RNA：circ_0011721与miR-519a表达呈负相关，二者在细胞浆发生共定位，进一步发现circ_0011721抑制GBM细胞自噬和化疗敏感性；生信分析表明该circRNA母基因MTF1启动子存在STAT3结合的位点，且STAT3能够促进MTF1 pre-mRNA的生成、上调circ_0011721的表达。由此我们提出假说:circ_0011721通过下调miR-519a与STAT3形成正反馈环路来抑制GBM细胞自噬，从而促进GBM耐药。本项目拟进一步通过体内、体外实验阐明circ_0011721-miR-519a-STAT3通路形成的机制，从而为逆转TMZ耐药提供新的靶点。

胶质母细胞瘤(GBM)的化疗过程中替莫唑胺(TMZ)耐药与自噬密切相关。前期研究中已发现了一个调控自噬和替莫唑胺化疗耐药的新通路:miR-519a/STAT3/BCL2 通路，但miR-519a上游调控机制尚未明确。本项目筛选到环状RNA:circ_0011721和miR-519a表达呈负相关，进一步发现二者在胞浆共定位,且circ_0011721抑制GBM细胞自噬；生信分析表明circ_0011721母基因MTF1启动子区存在与STAT3发生结合的位点,且STAT3能够促进 MTF1_pre-mRNA 的生成。我们通过CCK8、平板克隆、EDU实验、流式细胞术实验验证了在U87MG耐药株中，circ_0011721 促进GBM细胞替莫唑胺化疗耐药。通过WesternBlot、细胞转染LC3B-GFP质粒与电镜实验证实circ_0011721抑制TMZ诱导的自噬，并且进一步明确了自噬介导了circ_0011721诱导的化疗敏感性降低。通过双荧光素酶报告基因实验、生物素标记的核酸捕获实验、Ago2 免疫沉淀实验验证了circ_0011721和miR-519a结合并可以竞争miR-519a结合STAT3。通过 GTRD 在线分析工具寻找到 circ_0011721 宿主基因 MTF1上游2000kb的启动子区。通过染色质免疫共沉淀(ChIP-qPCR)检测STAT3可以通过MTF1启动子影响circ_0011721的表达。克隆 MTF1上游2000kb的启动子，并通过酶切及链接转移到 pGL3 vector 中行双荧光素酶报告基因实验(luciferase reporter assay)进一步验证了STAT3在转录水平调控 circ_0011721的表达增高。对含有 MTF1 启动子的 pGL3 质粒进行特异性点突变，寻找并确认了STAT3 在MTF1 上的特异结合的位点。同时，借助该项目的资助，我们也探讨了铁死亡介导胶质母细胞瘤TMZ化疗敏感性的机制研究，发现DRD2拮抗剂氟哌啶醇介导自噬诱导的铁死亡，通过促进胶质母细胞瘤内质网应激增加替莫唑胺敏感性。总之，本研究的结果表明：circ_0011721通过circ_0011721-miR-519a-STAT3正反馈调节通路在自噬和 GBM 替莫唑胺耐药中的关键作用。为胶质母细胞的化疗、预测提供了新的靶点。