Id1 knockdown in primary human glioma stem cells

Abnormal activation of stemness factors is a crucial signature of cancer stem cells (CSCs), a highly tumorigenic subpopulation in malignant tumors. However, it is unclear whether multi-signaling pathways are activated in CSCs, as like normal stem cells. I would like to report that an inhibitor of differentiation 1 (ID1) activates intracellular multi-signaling involved in proliferation, genesis, and maintenance of glioma stem cells (GSCs) by suppression of Cullin3, an E3 ubiquitin ligase that degrades Cyclin E and components of SHH and WNT signaling. ID1 inhibits BMP-dependent differentiation of GSCs by activation of BMPR2-targeting miR17/20a. ID1HIGH-Cullin3LOW signature correlates with a poor prognosis of GBM patients with a significant association to gene signatures enriched in EGF, WNT, SHH, and BMP signaling. Combinational inhibition of GSC intracellular multi-signaling network increases tumor-bearing mice survival. These results provide insights on molecular and cellular basis of GSC biology, and also suggest necessity of multi-signaling inhibition for GSCs therapy. Two human primary glioma stem cells (GSCs) such as GSC2 and GSC8 were isolated from two individual primary human glioma specimens. The GSCs were directly transfected with pSuper-GFP-ID1-shRNA and pSuper-GFP-Scrambled-shRNA using FuGENE 6 reagent (Roche). The RNA extraction in these cells was used to analyze gene expression.

干细胞干性因子的异常激活是癌症干细胞（CSCs）的关键特征，后者是恶性肿瘤中具有高致瘤性的细胞亚群。目前尚不明确，CSCs是否如正常干细胞一样存在多条信号通路的激活。本研究报道，分化抑制因子1（ID1）通过抑制Cullin3——一种可降解细胞周期蛋白E以及音猬因子（SHH）和WNT信号通路组分的E3泛素连接酶——激活胶质瘤干细胞（GSCs）增殖、发生与维持相关的胞内多条信号通路。ID1通过激活靶向骨形态发生蛋白受体2（BMPR2）的miR17/20a，抑制GSCs的骨形态发生蛋白（BMP）依赖型分化。ID1高表达-Cullin3低表达（ID1HIGH-Cullin3LOW）特征与胶质母细胞瘤（GBM）患者的不良预后显著相关，且与富集于表皮生长因子（EGF）、WNT、SHH及BMP信号通路的基因特征存在显著关联。联合抑制GSC胞内多信号网络可提高荷瘤小鼠的生存率。上述研究结果为阐明GSC生物学的分子与细胞基础提供了新的见解，同时也提示针对GSCs的治疗需采用多信号通路抑制策略。本研究从2例独立的原发性人脑胶质瘤标本中分离得到2株人原代胶质瘤干细胞（GSCs），即GSC2与GSC8。采用FuGENE 6转染试剂（罗氏）将pSuper-GFP-ID1-shRNA与pSuper-GFP-Scrambled-shRNA直接转染至上述GSCs中。提取上述细胞的RNA用于基因表达分析。