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.

胶质母细胞瘤（Glioblastoma）是最常见的恶性原发性脑肿瘤。目前临床相关的生物标志物仅局限于异柠檬酸脱氢酶（isocitrate dehydrogenase, IDH）1或2基因突变，以及O6-甲基鸟嘌呤DNA甲基转移酶（O6-methylguanine DNA methyltransferase, MGMT）启动子甲基化。长链非编码RNA（long non-coding RNA, lncRNA）的异常改变可能参与胶质母细胞瘤的发病机制，并有望成为新型生物标志物。本研究针对HOXA转录反义RNA、髓系特异性1（HOXA Transcript Antisense RNA, Myeloid-Specific 1, HOTAIRM1）的临床意义，在多组胶质母细胞瘤基因表达数据集中展开分析，探究其与患者预后、IDH突变及MGMT启动子甲基化状态的关联。研究通过体外及体内实验，对HOTAIRM1在胶质母细胞瘤生物学行为及放射抵抗中的作用进行了表征。我们鉴定HOTAIRM1为一种候选lncRNA，其表达上调与胶质母细胞瘤患者更短的总生存期显著相关，且该相关性独立于IDH突变与MGMT启动子甲基化状态。胶质母细胞瘤细胞系模型中，下调HOTAIRM1的表达可一致地降低细胞活力、抑制侵袭性生长，并削弱集落形成能力。整合蛋白质基因组学分析及活性氧（reactive oxygen species, ROS）水平检测结果显示，下调HOTAIRM1会导致线粒体功能受损及ROS水平升高。下调HOTAIRM1可降低谷氨酰胺转移酶2（transglutaminase 2, TGM2）的表达，而TGM2是与线粒体功能相关的候选蛋白；敲低TGM2可模拟下调HOTAIRM1在胶质母细胞瘤细胞中产生的表型。此外，HOTAIRM1可在体外及体内调节胶质母细胞瘤细胞的放射敏感性。本研究数据支持HOTAIRM1作为胶质母细胞瘤生物学侵袭性、放射抵抗及不良预后的驱动因子。靶向HOTAIRM1或可成为一种极具前景的新型治疗策略。整体实验设计：为实现HOTAIRM1的瞬时敲低，转染前一日将T98G、LN-229或U251胶质母细胞瘤细胞以每孔100,000个细胞的密度接种于6孔板中。通过使用HOTAIRM1特异性siPOOLs（#100506311, siTOOLs Technology, Planegg, Martinsried, Germany）及非靶向阴性对照siPOOLs（阴性对照siPOOL 5 nmol, siPOOL Technology），分别实现HOTAIRM1的特异性敲低及对应的非靶向对照处理。siPOOLs的转染采用Lipofectamine RNAiMAX Transfection Reagent（#13778150, Thermo Fischer Scientific）完成。具体操作如下：将7.5 μL RNAiMAX转染试剂稀释于125 μL Opti-MEM（#31985062, Thermo Fischer Scientific）中，随后将其与含有0.5 μL（5 pM）siPOOLs的125 μL Opti-MEM混合并加入孔板。转染72小时后收集细胞。