The long non-coding RNA HOTAIRM1 promotes tumor aggressiveness and radiotherapy resistance in glioblastoma
Glioblastoma is the most common malignant primary brain tumor. Clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutation and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNA (lncRNA) alterations may contribute to glioblastoma pathogenesis and potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was analyzed in multiple glioblastoma gene expression data sets for associations with prognosis and IDH mutation and MGMT promoter methylation status. The role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, less invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses and determination of reactive oxygen species (ROS) levels revealed impaired mitochondrial function and increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2) as a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach. Overall design: For transient knock-down of HOTAIRM1, T98G, LN-229 or U251 glioblastoma cells were seeded (100,000 cells per well) into 6-well plates the day before knock-down. Specific knock-down of HOTAIRM1 and the corresponding non-target negative controls was achieved by using HOTAIRM1-specific siPOOLs (#100506311, siTOOLs Technology, Planegg, Martinsried, Germany), siTOOLs Technology) and non-target siPOOLs (Neg. control siPOOL 5 nmol, siPOOL Technology), respectively. Transfection of siPOOLs was performed using Lipofectamine RNAiMAX Transfection Reagent (#13778150, Thermo Fischer Scientific). Briefly, 7.5 µl RNAiMAX Transfection Reagent was diluted in 125 µl Opti-MEM (#31985062, Thermo Fischer Scientific) pipetted into a well with 0.5 µl (5 pM) siPOOL in 125 µl Opti-MEM. Cells were harvested 72 hours post transfection.