Investigation of Notch-inhibiting microRNAs in glioma

描述（申请人提供）：胶质瘤是最常见、最致命的脑肿瘤，对放疗和化疗具有高度耐药性。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至关重要。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。在现在提交的后续工作中，我们发现胶质瘤中的其他关键途径被假定的肿瘤抑制因子microRNA-7所抑制。microrna是一种小的非编码rna，最近发现可以下调人类基因的一个大子集，有证据表明一些microrna具有强大的致癌或肿瘤抑制功能。我们对Notch通路和microRNA的研究使我们评估是否可能存在与Notch通路相反的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通路中有几个预测靶点，并抑制Notch活性，显示相对于正常大脑，胶质瘤中的表达降低，转染胶质瘤细胞后抑制活力和侵袭性。在这个应用中，我们建议系统研究microRNAs -7和-326作为notch抑制肿瘤抑制因子，具有治疗胶质瘤的潜力。在Aim #1中，我们将使用免疫印迹和3'-UTR荧光素酶报告器来验证哪些预测的Notch通路成员确实是这些microrna直接靶向的。此外，我们将确定microrna -7和-326抑制剂是否可以增加Notch活性和Notch通路靶点的表达。为了进一步研究这些microrna在Aim #2中的下调，我们将在人类胶质瘤和正常脑样本中量化这些microrna的各种形式的表达，以及它们与Notch活性和靶向Notch通路蛋白水平的相关性。我们还将在人类胶质瘤和正常大脑中进行全局microRNA分析，以确认这些microRNA的下调是选择性的。在Aim #3中，将评估转染microRNA- 7和microRNA-326到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，标准的癌细胞系可能不能很好地代表原来的肿瘤，而胶质瘤和其他癌症可能更准确地由肿瘤内一个小的干细胞样亚群生长出来的细胞系来模拟。据推测，这种干细胞样部分负责肿瘤的发生和肿瘤的再生，并且它们对标准治疗具有高度抗性。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肿瘤干细胞系生长、分化和侵袭性的影响。该实验的成功完成将确定microRNA-7和microRNA-326作为Notch通路的新型内源性抑制剂，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，并可能与发展和其他癌症相关。

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.