Investigation of Notch-inhibiting microRNAs in glioma

描述(申请人提供)：胶质瘤是最常见和最致命的脑肿瘤，对放射和化疗具有高度抵抗力。为了开发更有效的治疗方法，更好地了解这种癌症的分子机制是至关重要的。我们之前报道了Notch在胶质瘤中的致癌作用，Notch是干细胞维持和细胞命运决定的关键途径。在随后提交的工作中，我们发现胶质瘤中的其他关键通路受到一种假定的肿瘤抑制因子microRNA-7的抑制。MicroRNAs是最近发现的一种小的非编码RNA，可以下调人类基因的一大部分，有证据表明，一些microRNAs具有强大的致癌或肿瘤抑制功能。我们对Notch途径和microRNA的研究使我们评估了是否存在与Notch途径相反的microRNA在胶质瘤中发挥重要作用。初步研究发现了两个有希望的候选者，microRNA-7和microRNA-326。两者都在Notch通路中有几个预测的靶点，并抑制Notch的活性，在人脑胶质瘤中显示出相对于正常脑组织的表达减少，并在转染到胶质瘤细胞后抑制活性和侵袭力。在这一应用中，我们建议系统地研究microRNAs-7和-326作为Notch抑制肿瘤抑制因子对胶质瘤的治疗潜力。在目标1中，我们将使用免疫印迹和3‘-UTR荧光素酶报告来验证哪些预测的Notch途径成员确实是这些microRNAs的直接靶标。此外，我们将确定microRNAs-7和-326的抑制剂是否可以增加Notch活性和Notch途径靶标的表达。为了进一步研究这些microRNAs在AIM#2中的下调，这些microRNAs的不同形式在人脑胶质瘤和正常脑样本中的表达将被量化，它们的水平与Notch活性和靶向Notch途径蛋白的水平相关。我们还将在人脑胶质瘤和正常脑中进行全局microRNA分析，以确认这些microRNAs的下调是有选择性的。在目标#3中，将评估将microRNA-7和microRNA-326导入已建立的胶质瘤系的表型效应，并确定Notch抑制对这些效应的贡献。然而，最近的证据表明，标准的癌细胞株可能不能很好地代表原始肿瘤，而胶质瘤和其他癌症可能更准确地由肿瘤内一个类似干细胞的小亚群生长出来的株系模拟。据推测，这种干细胞样组分与肿瘤的发生和再生长有关，而且它们对标准治疗具有高度抵抗力。因此，我们将在AIM#4中确定microRNA-7和microRNA-326在体外和原位异种移植小鼠模型中对胶质瘤干细胞系生长、分化和侵袭力的影响。拟议实验的成功完成将确立microRNA-7和microRNA-326作为Notch途径的新型内源性抑制物，在胶质瘤中具有肿瘤抑制作用，对神经肿瘤学具有重要意义，并可能与发育和其他癌症相关。

DESCRIPTION (provided by applicant): Glioma is the most common and most lethal brain tumor, highly resistant to radiation and chemotherapy. To develop more effective treatments, it is vital to better understand the molecular mechanisms of this cancer. We previously reported in gliomas an oncogenic role for Notch, a critical pathway in stem cell maintenance and cell fate determination. In subsequent work now submitted, we showed that other key pathways in gliomas are inhibited by a putative tumor suppressor, microRNA-7. microRNAs are small non-coding RNAs recently found to down-regulate a large subset of human genes, and evidence suggests that some microRNAs have powerful oncogenic or tumor suppressor functions. Our investigation of both the Notch pathway and microRNA led us to assess whether there might be microRNAs opposing the Notch pathway with significant roles in gliomas. Preliminary studies revealed two promising candidates, microRNA-7 and microRNA-326. Both have several predicted targets in the Notch pathway and suppress Notch activity, show decreased expression in human gliomas relative to normal brain, and inhibit viability and invasiveness following transfection into glioma cells. In this application, we propose the systematic investigation of microRNAs -7 and -326 as Notch-inhibiting tumor suppressors with therapeutic potential for glioma. In Aim #1, we will use immunoblotting and 3'-UTR luciferase reporters to validate which of the predicted Notch pathway members are indeed directly targeted by these microRNAs. Additionally, we will determine whether inhibitors of microRNAs-7 and -326 can increase Notch activity and expression of Notch pathway targets. To further investigate down-regulation of these microRNAs in Aim #2, expression of the various forms of these microRNAs will be quantified in human glioma and normal brain samples and their levels correlated to Notch activity and to levels of targeted Notch pathway proteins. We will also perform global microRNA profiling in human gliomas and normal brain to confirm that down-regulation of these microRNAs is selective. In Aim #3, the phenotypic effects of transfecting microRNA- 7 and microRNA-326 into established glioma lines will be evaluated and the contribution of Notch inhibition to these effects determined. Recent evidence has suggested that standard cancer cell lines may be poorly representative of the original tumors, however, and that gliomas and other cancers may be more accurately modeled by lines grown from a small stem cell-like subpopulation within the tumors. It is hypothesized that this stem cell-like fraction is responsible for tumorigenesis and regrowth of tumors, and they are highly resistant to standard therapies. We will therefore determine in Aim #4 the effects of microRNA-7 and microRNA-326 on growth, differentiation, and invasiveness in glioma tumor stem cell lines in vitro as well as in orthotopic xenograft mouse models. Successful completion of the proposed experiments will establish microRNA-7 and microRNA-326 as novel endogenous inhibitors of the Notch pathway with tumor suppressor roles in gliomas, with importance for neuro-oncology and likely relevance for development and for other cancers.