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Abstract：
BACKGROUND: Angiogenesis 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.
METHODS: The 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 .
RESULTS: We 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.
CONCLUSIONS: Our 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.
METHODS: The 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 .
RESULTS: We 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.
CONCLUSIONS: Our 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.
摘要：
背景：血管生成在神经胶质瘤的进展中起关键作用。已有研究表明,RNA结合蛋白(RBPs)与RNA相互作用,参与调控肿瘤的恶性行为。作为一种内源性非编码RNA,环状RNA（circularRNAs，circularRNAs）在各种癌症中异常表达，并参与包括血管生成在内的多种肿瘤发生。
方法：FUS的表达水平，circ_002136,miR-138-5p,使用定量实时PCR(qRT-PCR)和蛋白质印迹测定S0X13和SP0N2。使用脂质体3000试剂进行瞬时细胞转染。RNA结合蛋白免疫沉淀(RNA-IP)和RNA下拉测定用于检测FUS和circ_002136之间的相互作用。双荧光素酶报告基因检测系统检测circ_002136和miR-138-5p的结合位点,miR-138-5p和SOX13。染色质免疫沉淀（ChIP）测定用于检查转录因子SOX13与其靶蛋白之间的相互作用。
结果：我们证明了FUS或circ_002136的下调显著抑制了活力，U87胶质瘤暴露的内皮细胞（GECs）的迁移和管形成。miR-138-5p在GEC中下调,并且circ_002136在RNA诱导的沉默复合物(RISC)中功能性靶向miR-138-5p。抑制circ_002136，结合miR-138-5p的恢复，强烈地减少了GECs的血管生成。作为miR-138-5p的靶基因,SOX13在GECs中过表达，并被证明参与circ_002136和miR-138-5p介导的神经胶质瘤血管生成。此外,我们发现SOX13与SPON2启动子直接相关并被激活，从而在转录水平上调SPON2的表达。SPON2的敲除抑制GECs中的血管生成。更重要的是,SOX13激活FUS启动子并增加其表达，形成一个反馈回路。
结论：我们的数据表明，FUS/circ_002136/miR-138-5p/SOX13的反馈回路在神经胶质瘤血管生成的调节中起着至关重要的作用。这也为联合神经胶质瘤治疗提供了潜在的靶标和替代策略。
方法：FUS的表达水平，circ_002136,miR-138-5p,使用定量实时PCR(qRT-PCR)和蛋白质印迹测定S0X13和SP0N2。使用脂质体3000试剂进行瞬时细胞转染。RNA结合蛋白免疫沉淀(RNA-IP)和RNA下拉测定用于检测FUS和circ_002136之间的相互作用。双荧光素酶报告基因检测系统检测circ_002136和miR-138-5p的结合位点,miR-138-5p和SOX13。染色质免疫沉淀（ChIP）测定用于检查转录因子SOX13与其靶蛋白之间的相互作用。
结果：我们证明了FUS或circ_002136的下调显著抑制了活力，U87胶质瘤暴露的内皮细胞（GECs）的迁移和管形成。miR-138-5p在GEC中下调,并且circ_002136在RNA诱导的沉默复合物(RISC)中功能性靶向miR-138-5p。抑制circ_002136，结合miR-138-5p的恢复，强烈地减少了GECs的血管生成。作为miR-138-5p的靶基因,SOX13在GECs中过表达，并被证明参与circ_002136和miR-138-5p介导的神经胶质瘤血管生成。此外,我们发现SOX13与SPON2启动子直接相关并被激活，从而在转录水平上调SPON2的表达。SPON2的敲除抑制GECs中的血管生成。更重要的是,SOX13激活FUS启动子并增加其表达，形成一个反馈回路。
结论：我们的数据表明，FUS/circ_002136/miR-138-5p/SOX13的反馈回路在神经胶质瘤血管生成的调节中起着至关重要的作用。这也为联合神经胶质瘤治疗提供了潜在的靶标和替代策略。
