FUS/circ_002136/miR-138-5p/SOX13 feedback loop regulates angiogenesis in Glioma
FUS/circ_002136/miR-138-5p/SOX13 feedback loop regulates angiogenesis in Glioma
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DOI：
10.1186/s13046-019-1065-7
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发表时间：
2019-02-08
影响因子：
11.3
通讯作者：
Xue, Yixue
中科院分区：
文献类型：
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作者：
He, Zhenwei;Ruan, Xuelei;Xue, Yixue
背景血管生成在神经胶质瘤的进展中起着关键作用。此前的研究表明，RNA结合蛋白（RBP）与RNA相互作用，参与肿瘤恶性行为的调节。环状RNA（circRNA）作为一类内源性非编码RNA，在多种癌症中异常表达，并参与包括血管生成在内的多种肿瘤发生。方法采用实时定量PCR（qRT-PCR）和蛋白质印迹法测定FUS、circ_002136、miR-138-5p、SOX13和SPON2的表达水平。使用 Lipofectamine 3000 试剂进行瞬时细胞转染。 RNA 结合蛋白免疫沉淀 (RNA-IP) 和 RNA Pull-down 测定用于检测 FUS 和 circ_002136 之间的相互作用。采用双荧光素酶报告基因检测系统检测circ_002136与miR-138-5p、miR-138-5p和SOX13的结合位点。使用染色质免疫沉淀 (ChIP) 检测转录因子 SOX13 与其靶蛋白之间的相互作用。结果我们证明 FUS 或 circ_002136 的下调显着抑制 U87 胶质瘤暴露的内皮细胞 (GEC) 的活力、迁移和管形成。 MiR-138-5p 在 GEC 中下调，circ_002136 在 RNA 诱导的沉默复合物 (RISC) 中功能性靶向 miR-138-5p。 circ_002136 的抑制与 miR-138-5p 的恢复相结合，显着减少了 GEC 的血管生成。作为 miR-138-5p 的靶基因，SOX13 在 GEC 中过表达，并被证明参与 circ_002136 和 miR-138-5p 介导的胶质瘤血管生成。此外，我们发现SOX13直接与SPON2启动子相关并激活SPON2启动子，从而在转录水平上调SPON2的表达。 SPON2 的敲低抑制了 GEC 中的血管生成。更重要的是，SOX13激活FUS启动子并增加其表达，形成反馈环。结论我们的数据表明FUS/circ_002136/miR-138-5p/SOX13的反馈环在胶质瘤血管生成的调节中发挥着至关重要的作用。这也为神经胶质瘤联合治疗提供了潜在的靶点和替代策略。
BackgroundAngiogenesis plays a critical role in the progression of glioma. Previous studies have indicated that RNA-binding proteins (RBPs) interact with RNAs and participate in the regulation of the malignant behaviors of tumors. As a type of endogenous non-coding RNAs, circular RNAs (circRNAs) are abnormally expressed in various cancers and are involved in diverse tumorigeneses including angiogenesis.MethodsThe expression levels of FUS, circ_002136, miR-138-5p, SOX13, and SPON2 were determined using quantitative real-time PCR (qRT-PCR) and western blot. Transient cell transfection was performed using the Lipofectamine 3000 reagent. The RNA-binding protein immunoprecipitation (RNA-IP) and the RNA pull-down assays were used to detect the interaction between FUS and circ_002136. The dual-luciferase reporter assay system was performed to detect the binding sites of circ_002136 and miR-138-5p, miR-138-5p and SOX13. The chromatin immunoprecipitation (ChIP) assays were used to examine the interactions between transcription factor SOX13 and its target proteins .ResultsWe demonstrated that down-regulation of FUS or circ_002136 dramatically inhibited the viability, migration and tube formation of U87 glioma-exposed endothelial cells (GECs). MiR-138-5p was down-regulated in GECs and circ_002136 functionally targeted miR-138-5p in an RNA-induced silencing complex (RISC). Inhibition of circ_002136, combined with the restoration of miR-138-5p, robustly reduced the angiogenesis of GECs. As a target gene of miR-138-5p, SOX13 was overexpressed in GECs and was proved to be involved in circ_002136 and miR-138-5p-mediated angiogenesis in gliomas. In addition, we found that SOX13 was directly associated with and activated the SPON2 promoter, thereby up-regulating the expression of SPON2 at the transcriptional level. Knockdown of SPON2 suppressed the angiogenesis in GECs. More important, SOX13 activated the FUS promoter and increased its expression, forming a feedback loop.ConclusionOur data suggests that the feedback loop of FUS/circ_002136/miR-138-5p/SOX13 played a crucial role in the regulation of angiogenesis in glioma. This also provides a potential target and an alternative strategy for combined glioma therapy.