ABSTRACT

This study aims to explore the molecular mechanism by which HAS2-AS1 acts as a ceRNA to promote the invasion and migration of glioma cells, which will provide a novel potential target for the targeted therapy of glioma. Gene expression profiles and corresponding clinical data were accessed from the TCGA_LGG and TCGA_GBM databases and then differential analysis was conducted using the “edgeR” package. miRDB, miRTarBase and TargetScan databases were employed to predict target genes and sequentially a ceRNA network was constructed. Quantitative real-time PCR was performed to detect gene expression in glioma cells. Transwell assay was operated to assess cell migratory and invasive abilities. Western blot was conducted to evaluate the protein expression. Dual-luciferase reporter assay and RNA immunoprecipitation experiment were performed to validate the targeting relationship between genes. HAS2-AS1 was markedly upregulated in glioma, and the overall survival time of patients with high HAS2-AS1 expression was significantly shorter than that of patients with low one. Silencing HAS2-AS1 inhibited the migration and invasion of glioma cells, while overexpressing HAS2-AS1 produced opposite results. miR-137 was validated as a direct target of and negatively regulated by HAS2-AS1. Further exploration of the downstream target gene indicated that EZH2 competed with HAS2-AS1 to interact with miR-137. Suppressing miR-137 or up-regulating EZH2 reversed the impact of HAS2-AS1 knockdown on glioma cell invasion and migration. HAS2-AS1 regulates EZH2 by sponging miR-137 for the migratory and invasive abilities of glioma cells, which provides a new idea for exploring metastasis mechanism of glioma.

摘要

本研究旨在探索HAS2-AS1作为ceRNA促进胶质瘤细胞侵袭和迁移的分子机制，为胶质瘤靶向治疗提供新的潜在靶点。从 TCGA_LGG 和 TCGA_GBM 数据库中获取基因表达谱和相应的临床数据，然后使用“edgeR”包进行差异分析。使用 miRDB、miRTarBase 和 TargetScan 数据库来预测靶基因，并依次构建 ceRNA 网络。进行定量实时 PCR 以检测神经胶质瘤细胞中的基因表达。操作 Transwell 测定以评估细胞迁移和侵袭能力。进行蛋白质印迹以评估蛋白质表达。进行双荧光素酶报告基因测定和RNA免疫沉淀实验以验证基因之间的靶向关系。HAS2-AS1在胶质瘤中显着上调，HAS2-AS1高表达患者的总生存时间明显短于低表达患者。沉默 HAS2-AS1 会抑制胶质瘤细胞的迁移和侵袭，而过表达 HAS2-AS1 会产生相反的结果。miR-137 被证实为 HAS2-AS1 的直接靶标并受其负调控。对下游靶基因的进一步探索表明，EZH2 与 HAS2-AS1 竞争与 miR-137 相互作用。抑制 miR-137 或上调 EZH2 可逆转 HAS2-AS1 敲低对胶质瘤细胞侵袭和迁移的影响。