miR-340通过Notch信号通路介导GSCs的“干性”抑制

The stem-like cell function in glioma stem cells (GSCs) is the key reason to account for the difficulty to treat with Glioblastoma Multiforme (GBM). In our early study using microRNA microarrays, we found that miR-340 expression was decreased significantly in all GSCs, Preliminary experiment illustrates that miR-340 overexpression in GSCs decreases Notch expression and suppresses the stem-like cell function. Based upon these about miR-340, we hypothesize that miR-340 suppresses the stem-like cell function of GSCs by regulating Notch signaling pathway. In the following study we will use GSCs isolated and cultured with serum-free cell culture technique and GBM orthotopic animal model. The activation of signal pathway, cellular morphology, proliferative, invasive and migratory properties and chemotherapy resistance are monitored while miR-340 overexpression and Notch knockdown using RNAi. By comparing the similarities and differences of the miR-340 overexpression and the Notch knockdown in the suppression of tumor growth, we prove that miR-340 suppresses the stem-like cell function of glioma stem cells by regulating Notch signaling pathway theoretically and experimentally. On this basis, the in situ hybridization technology is used to detect miR-340 level in GBM samples, and construct the risk score model to demonstrate whether miR-340 can be used as a prognostic predictor for malignant glioma. This study will reveal the pathogenesis of GBM from an uncharted viewpoint of mir-340, and provide new ideas for the diagnosis and treatment of malignant glioma.

胶质瘤干细胞（GSCs） “干性”是GBM难以治愈的根本原因，我们使用miRNA microarrays筛选出miR-340在GSCs中表达显著下降，预实验结果过表达miR-340可以抑制Notch蛋白表达，且GSCs“干性”受到抑制。为此，我们提出miR-340通过Notch信号通路介导GSCs的“干性”抑制假说，通过无血清技术分离培养的GSCs和小鼠原位肿瘤模型，分别上调miR-340或干扰Notch蛋白检测Notch信号通路激活及GSCs形态学、自我更新、侵袭迁移、耐药能力改变， 比较两种方法介导的抑瘤作用，明确miR-340通过Notch信号通路介导GSCs“干性”抑制。利用原位杂交技术检测miR-340表达水平，构建危险评分数学模型，论证miR-340是否可以作为GBM预后预测因子。本研究从miR-340这个全新视点为揭示GBM的发病机制奠定基础，为恶性胶质瘤的诊疗提供新的思路。

在本项目中，我们提出miR-340通过Notch信号通路介导GSCs的“干性”抑制假说；拟使用生物信息学，分子生物学和细胞生物学的技术手段，明确miR-340可以通过靶向Notch的3’UTR来抑制其表达，从而影响胶质瘤干细胞的“干性”；拟通过胶质瘤原位移殖瘤模型，证明miR-340在体内对胶质瘤干细胞的“干性”的抑制作用；拟通过原位杂交技术的使用和危险评分数学模型的建立，检测临床GBM样本中miR-340 与患者生存期、预后及治疗疗效之间的关系，回归分析论证miR-340 是否可以作为临床恶性胶质瘤预后预测因子。 .研究的结果如下：microRNA microarrays和 qRT-PCR确认了miR-340在原代分离培养的GSCs中的表达水平降低；使用合成的miR-340类似物Mimics-miR-340上调miR-340后，发现了能够抑制GSCs的干性；明确了miR-340能够直接负调控Notch，并由此实现对GSCs“干性”的抑制；在移殖瘤模型中原位注射miR-340的agomir，验证了miR-340在体内抑制了GSCs“干性”。利用原位杂交技术，发现了miR-340 水平与患者预后存在负相关关系，确定了miR-340 可以作为临床恶性胶质瘤预后预测因子。.我们的研究结果将有助于人们对 microRNA在GBM发生发展中的作用机制的认识，也为选择miR-340作为临床恶性胶质瘤的预后预测因子提供依据。