Chromatin states in glioblastoma stem cells (HOXA-AS2 depletion)

Glioblastoma multiform account for about half of all gliomas and are the most deadly and aggressive forms. Its therapeutic resistance and tumor relapse rely on a subpopulation of cells, the so-called Glioma-stem Cells (GSCs). Here, we investigated for the role of the long non-coding RNA HOXA-AS2 in GSC biology by conducting descriptive and functional analyses of glioma samples classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found that HOXA-AS2 is overexpressed only in aggressive (IDHwt) glioma and GSC. Sh-RNA-based depletion of HOXA-AS2 affects GSC both at the cellular and molecular levels with a decrease in proliferation and altered expression of several hundreds of their genes. Integrative analysis revealed that these changes is expression are not associated to changes in DNA methylation or chromatin signature at the promoter of most deregulated genes following HOXA-AS2 silencing in GSC, supporting a post-transcriptional regulation. In addition, transcription factor motif enrichment and correlation analyses sustained that HOXA-AS2 affect, directly or indirectly, expression of key transcription factors of GCS biology, including E2F8, E2F1, STAT1 and ATF3 to, in fine, contributes to their pathological status by promoting proliferation and modulating the inflammation pathway of Glioma Stem Cell. Overall design: HOXA-AS2 was depleted in human glioblastoma stem cells, and ChIP-seq was performed using anti-H3K27ac, anti-H3K4me3, and anti-H3K27me3 antibodies.

多形性胶质母细胞瘤（Glioblastoma multiform）约占所有胶质瘤的半数，是致死性最强、侵袭性最高的胶质瘤亚型。其治疗抵抗与肿瘤复发依赖于一类细胞亚群——即所谓的胶质瘤干细胞（Glioma-stem Cells, GSCs）。本研究围绕长链非编码RNA HOXA-AS2在胶质瘤干细胞生物学中的作用展开探究，通过根据异柠檬酸脱氢酶（isocitrate dehydrogenase, IDH1）基因突变状态分类的胶质瘤样本，以及胶质瘤干细胞开展描述性与功能分析。研究发现，HOXA-AS2仅在侵袭性（IDH野生型，IDHwt）胶质瘤及胶质瘤干细胞中呈高表达。基于短发夹RNA（Sh-RNA）的HOXA-AS2敲除可在细胞与分子层面影响胶质瘤干细胞：不仅使细胞增殖能力下降，还可改变数百个相关基因的表达谱。整合分析显示，在胶质瘤干细胞中敲低HOXA-AS2后，绝大多数失调基因的启动子区域未出现DNA甲基化或染色质特征的显著变化，提示其调控机制为转录后调控。此外，转录因子基序富集分析与相关性分析证实，HOXA-AS2可直接或间接影响胶质瘤干细胞生物学关键转录因子的表达，包括E2F8、E2F1、STAT1及ATF3，最终通过促进增殖、调控胶质瘤干细胞的炎症通路，参与其病理状态的维持。整体实验设计：本研究在人胶质母细胞瘤干细胞中敲低HOXA-AS2，并使用抗H3K27ac、抗H3K4me3及抗H3K27me3抗体完成染色质免疫沉淀测序（ChIP-seq）。