Investigation of Notch-inhibiting microRNAs in glioma

描述（由申请人提供）：胶质瘤是最常见和最致命的脑肿瘤，对放疗和化疗具有高度抗性。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。我们先前报道了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推定的肿瘤抑制因子microRNA-7抑制。microRNA是最近发现的下调人类基因的大子集的小的非编码RNA，并且证据表明一些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，其在胶质瘤中具有重要作用。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途径中具有几个预测的靶点并抑制Notch活性，相对于正常脑，在人脑胶质瘤中显示出降低的表达，并在转染到胶质瘤细胞中后抑制活力和侵袭力。在本申请中，我们提出了microRNA-7和-326作为Notch抑制性肿瘤抑制剂的系统研究，其具有治疗胶质瘤的潜力。在目标#1中，我们将使用免疫印迹和3 '-UTR荧光素酶报告基因来验证哪些预测的Notch途径成员确实被这些microRNA直接靶向。此外，我们将确定microRNA-7和-326的抑制剂是否可以增加Notch活性和Notch途径靶点的表达。为了进一步研究目标#2中这些微小RNA的下调，将在人神经胶质瘤和正常脑样品中定量这些微小RNA的各种形式的表达，并且将其水平与Notch活性和靶向Notch途径蛋白的水平相关联。我们还将在人类神经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。在目标#3中，将评估将微小RNA- 7和微小RNA-326转染到建立的神经胶质瘤系中的表型效应，并确定Notch抑制对这些效应的贡献。然而，最近的证据表明，标准癌细胞系可能不能很好地代表原始肿瘤，并且神经胶质瘤和其他癌症可以通过从肿瘤内的小干细胞样亚群生长的细胞系更准确地建模。假设这种干细胞样部分负责肿瘤发生和肿瘤再生长，并且它们对标准疗法具有高度抗性。因此，我们将在目标#4中确定microRNA-7和microRNA-326对体外神经胶质瘤肿瘤干细胞系以及原位异种移植小鼠模型中的生长、分化和侵袭性的影响。成功完成拟议的实验将建立microRNA-7和microRNA-326作为Notch途径的新型内源性抑制剂，在神经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。公共卫生相关性：神经胶质瘤是最常见和致命的脑肿瘤。它们对放疗和化疗具有抗性，因此研究其分子机制并开发新的治疗策略至关重要。最近的证据表明，新发现的称为microRNA的非编码RNA可以在胶质瘤和其他癌症中发挥致癌或肿瘤抑制作用。每一种微小RNA都能抑制许多靶基因的翻译，其中一些还能调节关键的癌症通路。在这项提案中，我们将研究两种microRNA作为肿瘤抑制剂和治疗胶质瘤继发于其抑制Notch途径，干细胞，细胞命运和癌症的中心途径。

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. PUBLIC HEALTH RELEVANCE: Gliomas are the most common and lethal brain tumors. They are resistant to radiation and chemotherapy, making it critical to dissect their molecular mechanisms and develop novel therapeutic strategies. Recent evidence has indicated that newly-discovered non-coding RNAs called microRNAs can play oncogenic or tumor suppressor roles in gliomas and other cancers. Each microRNA inhibits translation of numerous target genes, and a few regulate key cancer pathways. In this proposal, we will investigate two microRNAs as tumor suppressors and therapies in glioma secondary to their inhibition of the Notch pathway, a central pathway in stem cells, cell fate, and cancer.