Abstract

Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, 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, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), 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 both 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.

Highlights

  • Glioblastoma is the most malignant type of astrocytic glioma and the most common malignant primary brain tumor [1]

  • HOTAIRM1 knock-down sensitizes glioblastoma cells to radiation in vitro and in vivo Since radiation sensitivity has been associated with intracellular reactive oxygen species (ROS) levels [44] and since radiotherapy is an essential part of glioblastoma treatment, we investigated whether altered levels of HOTAIRM1 affect radiosensitivity of glioblastoma cells

  • Did we validate this finding in vitro, but our data indicate that high expression of HOTAIRM1 supports radioresistance of glioblastoma cells, which in turn may contribute to shorter patient survival as seen in our in vivo model

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Summary

INTRODUCTION

Glioblastoma is the most malignant type of astrocytic glioma and the most common malignant primary brain tumor [1]. 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. Previous studies have reported that HOTAIRM1 expression is increased in high-grade gliomas and in recurrent compared to primary glioblastomas [19, 20]. HOTAIRM1 has been shown to promote glioma growth and invasion through up-regulation of HOXA1 expression [21], through longplasmids (pMDL/pRRE, pRSV-Rev and pMD2.G) were transfected into HEK293T cells using polyethylenimine (#408727, PEI, Sigma-Aldrich). HOTAIRM1 has been proposed to promote glioma growth by acting as a sponge for several tumor suppressive miRNAs [24,25,26] We extend these findings by providing further clinical and functional evidence.

MATERIAL AND METHODS
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