RNA-seq analysis of glioblastoma stem cells where MEOX2 expression was knocked down by shRNAs. RNA-seq analysis of glioblastoma stem cells where MEOX2 expression was knocked down by shRNAs

The most widely accepted hypothesis about glioblastoma onset indicates glioblastoma stem-like cells (GSCs) as the cells of origin, also responsible for the high chemo- and radio-resistance of this tumor and of its recurrence. MEOX2 is a transcription factor overexpressed in glioblastoma, whose expression is negatively correlated to patients’ survival. Starting from our observation that MEOX2 expression is strongly enhanced in six GSC lines we tested, we performed shRNA-mediated knock-down experiments in two different GSC lines, and found that MEOX2 depletion inhibits their self-renewal and growth ability, and increases the apoptotic cell death. By a deep transcriptome analysis, we identified a core group of genes modulated in response to MEOX2 knock-down. Among these genes, the repressed ones are largely enriched in genes involved in the hypoxic response and in the glycolytic pathway, two strictly related pathways which contribute to the resistance of high grade gliomas to therapies. Overall design: Total transcriptome results of two different human glioblastoma stem cell (GSC) lines, BT273 and BT379, transduced with lentiviral vectors expressing shRNAs targeting MEOX2. Each GSC type transduced with one specific anti-MEOX2 shRNA was assayed in two biological replicates, and the results compared to the same GSC type transduced with a negative control vector, assayed in two biological replicates.

目前关于胶质母细胞瘤（glioblastoma）发病机制最广为接受的假说认为，胶质母细胞瘤干细胞样细胞（glioblastoma stem-like cells, GSCs）是该肿瘤的起源细胞，同时也是其具备高度化疗耐药、放疗抵抗特性以及发生复发的核心原因。MEOX2是一种在胶质母细胞瘤中过表达的转录因子，其表达水平与患者生存期呈显著负相关。基于前期在6株受试胶质母细胞瘤干细胞系中观察到的MEOX2表达显著上调现象，本研究在两株不同的胶质母细胞瘤干细胞系中开展了短发夹RNA（shRNA）介导的基因敲低实验，结果发现MEOX2基因沉默可抑制细胞的自我更新与增殖能力，并促进细胞凋亡。通过深度转录组分析，我们鉴定出了一组响应MEOX2基因敲低的核心调控基因。在这些基因中，受负调控的基因主要富集于缺氧应答与糖酵解通路——这两条紧密关联的通路正是高级别胶质瘤具备治疗抵抗能力的关键分子基础。

实验整体设计：本研究针对两株不同的人源胶质母细胞瘤干细胞系（GSCs）BT273与BT379，使用靶向MEOX2的短发夹RNA慢病毒载体进行转导，随后对其开展总转录组分析。每株经特异性抗MEOX2 shRNA转导的胶质母细胞瘤干细胞系均设置两组生物学重复，并将其测序结果与转导阴性对照载体的同株胶质母细胞瘤干细胞系（同样设置两组生物学重复）的测序结果进行对比。