Department of Neurobiology, School of Life Sciences, China Medical University, Shenyang, 110122, People's Republic of China.
Key Laboratory of Cell Biology, Ministry of Public Health of China, and Key Laboratory of Medical Cell Biology, Ministry of Education of China, China Medical University, Shenyang, 110122, People's Republic of China.
J Exp Clin Cancer Res. 2019 Feb 8;38(1):65. doi: 10.1186/s13046-019-1065-7.
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.
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 .
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.
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.
血管生成在神经胶质瘤的进展中起着关键作用。先前的研究表明，RNA 结合蛋白（RBPs）与 RNA 相互作用，并参与调节肿瘤的恶性行为。作为一种内源性非编码 RNA，circRNAs 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。
采用实时定量 PCR（qRT-PCR）和 Western blot 检测 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。采用 RNA 免疫沉淀（RNA-IP）和 RNA 下拉实验检测 FUS 与 circ_002136 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 与 SOX13 的结合位点。采用染色质免疫沉淀（ChIP）实验检测转录因子 SOX13 与其靶蛋白之间的相互作用。
我们发现下调 FUS 或 circ_002136 可显著抑制 U87 神经胶质瘤暴露的内皮细胞（GEC）的活力、迁移和管形成。miR-138-5p 在 GECs 中下调，circ_002136 在 RNA 诱导的沉默复合物（RISC）中靶向 miR-138-5p。抑制 circ_002136，结合恢复 miR-138-5p，可显著降低 GEC 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 介导的胶质瘤血管生成。此外，我们发现 SOX13 直接与 SPON2 启动子结合并激活该启动子，从而在转录水平上调 SPON2 的表达。敲低 SPON2 可抑制 GEC 的血管生成。更重要的是，SOX13 激活 FUS 启动子并增加其表达，形成反馈回路。
我们的数据表明，FUS/circ_002136/miR-138-5p/SOX13 的反馈环在调节神经胶质瘤中的血管生成中起着关键作用。这也为联合胶质瘤治疗提供了一个潜在的靶点和一种替代策略。
