背景:缺氧与包括胶质母细胞瘤(GBM)在内的许多癌症的不良预后相关。神经胶质瘤干细胞样细胞(GSC)通常位于缺氧区域,并作为疾病进展的储库。长非编码RNA(lncRNA)已经与GBM有关。然而,对调节GSC适应缺氧的lncRNAs了解甚少。这些lncRNAs的鉴定可能为在缺氧条件下靶向GSCs提供新的治疗策略。
方法:通过RNAseq鉴定缺氧诱导的GSCs中的lncRNAs。通过qPCR评估LUCAT1表达,RNAseq,北方印迹,GSC中的单分子FISH,在IvyGAP审讯,TCGA,CGGA数据库。LUCAT1被shRNA耗尽,CRISPR/Cas9和CRISPR/Cas13d。RNAseq,蛋白质印迹,免疫组织化学,co-IP,ChIP,ChIPseq,RNA免疫沉淀,进行了邻近连接试验以研究LUCAT1的作用机制。GSC生存能力,有限稀释试验,和原位GBM异种移植模型中的致瘤潜力进行评估,以评估消耗LUCAT1的功能后果.
结果:HIF1α和NRF2在缺氧条件下GSCs中诱导Lucat1的新同工型。LUCATl在GBM中的低氧区域中高度表达。机械上,LUCAT1与HIF1α及其共激活剂CBP形成复合物,以调节HIF1α靶基因表达和GSC对缺氧的适应。LUCAT1的耗尽损害了GSC的自我更新。在GBM异种移植模型中沉默LUCAT1降低肿瘤生长并延长小鼠存活。
结论:HIF1α-LUCAT1轴形成一个正反馈回路,在缺氧条件下扩增GSCs中的HIF1α信号。LUCAT1促进GSC自我更新和GBM肿瘤生长。LUCAT1是GBM的潜在治疗靶点。
BACKGROUND: Hypoxia is associated with poor prognosis in many cancers including glioblastoma (GBM). Glioma stem-like cells (GSCs) often reside in hypoxic regions and serve as reservoirs for disease progression. Long non-coding RNAs (lncRNAs) have been implicated in GBM. However, the lncRNAs that modulate GSC adaptations to hypoxia are poorly understood. Identification of these lncRNAs may provide new therapeutic strategies to target GSCs under hypoxia.
METHODS: lncRNAs induced by hypoxia in GSCs were identified by RNA-seq. Lung cancer-associated transcript-1 (LUCAT1) expression was assessed by qPCR, RNA-seq, Northern blot, single molecule FISH in GSCs, and interrogated in IvyGAP, The Cancer Genome Atlas, and CGGA databases. LUCAT1 was depleted by shRNA, CRISPR/Cas9, and CRISPR/Cas13d. RNA-seq, Western blot, immunohistochemistry, co-IP, ChIP, ChIP-seq, RNA immunoprecipitation, and proximity ligation assay were performed to investigate mechanisms of action of LUCAT1. GSC viability, limiting dilution assay, and tumorigenic potential in orthotopic GBM xenograft models were performed to assess the functional consequences of depleting LUCAT1.
RESULTS: A new isoform of Lucat1 is induced by Hypoxia inducible factor 1 alpha (HIF1α) and Nuclear factor erythroid 2-related factor 2 (NRF2) in GSCs under hypoxia. LUCAT1 is highly expressed in hypoxic regions in GBM. Mechanistically, LUCAT1 formed a complex with HIF1α and its co-activator CBP to regulate HIF1α target gene expression and GSC adaptation to hypoxia. Depletion of LUCAT1 impaired GSC self-renewal. Silencing LUCAT1 decreased tumor growth and prolonged mouse survival in GBM xenograft models.
CONCLUSIONS: A HIF1α-LUCAT1 axis forms a positive feedback loop to amplify HIF1α signaling in GSCs under hypoxia. LUCAT1 promotes GSC self-renewal and GBM tumor growth. LUCAT1 is a potential therapeutic target in GBM.