A miR-297/hypoxia/DGK-α axis regulating glioblastoma survival

• Published in: Neuro-oncology (Charlottesville, Va.) Vol. 15; no. 12; pp. 1652 - 1663
• Main Authors: Kefas, Benjamin, Floyd, Desiree H, Comeau, Laurey, Frisbee, Alyse, Dominguez, Charli, Dipierro, Charles G, Guessous, Fadila, Abounader, Roger, Purow, Benjamin
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.12.2013
• Subjects: 3' Untranslated Regions - genetics; Animals; Apoptosis; Basic and Translational Investigations; Blotting, Western; Brain Neoplasms - metabolism; Brain Neoplasms - mortality; Brain Neoplasms - pathology; Cell Movement; Cell Proliferation; Diacylglycerol Kinase - genetics; Diacylglycerol Kinase - metabolism; Flow Cytometry; Glioblastoma - metabolism; Glioblastoma - mortality; Glioblastoma - pathology; Humans; Hypoxia - genetics; Male; Mice; Mice, Inbred BALB C; Mice, SCID; MicroRNAs - genetics; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays; hypoxia; microRNA-297; apoptosis; cancer; glioblastoma; diacylglycerol kinase-alpha
• ISSN: 1522-8517; 1523-5866
• DOI: 10.1093/neuonc/not118

Despite advances in the treatment of the most aggressive form of brain tumor, glioblastoma, patient prognosis remains disappointing. This failure in treatment has been attributed to dysregulated oncogenic pathways, as observed in other tumors. We and others have suggested the use of microRNAs (miRs) as therapeutic tools able to target multiple pathways in glioblastoma. This work features PCR quantification of miRs and transient transfection of many glioblastoma cell lines with miRs, followed by cell number analysis, trypan blue staining, alamarBlue assay of cell viability, caspase-3/-7 activity assay, immunoblot of cleaved poly(ADP-ribose) polymerase and fluorescence activated cell sorting and imaging of apoptotic nuclei, cell invasion assays, MRIs of glioblastoma xenografts in mice using transiently transfected cells as well as posttumor treatment with lentiviral vector encoding miR-297, and analysis of miR-297 target diacylglycerol kinase (DGK)-α including immunoblot, 3'UTR luciferase activity, and rescue with DGK-α overexpression. Cell counts and DGK-α immunoblot were also analyzed in the context of hypoxia and with overexpression of heterogeneous ribonucleoprotein L (hnRNPL). We identified miR-297 as a highly cytotoxic microRNA in glioblastoma, with minimal cytotoxicity to normal astrocytes. miR-297 overexpression reduced in vitro invasiveness and in vivo tumor formation. DGK-α is shown to be a miR-297 target with a critical role in miR-297 toxicity. In addition, hypoxia and its mediator hnRNPL upregulated DGK-α and buffered the cytotoxic effects of miR-297. This work shows miR-297 as a novel and physiologic regulator of cancer cell survival, largely through targeting of DGK-α, and also indicates that hypoxia ameliorates miR-297 toxicity to cancer cells.