Apoptosis Effectors Targeted By Hedgehog-Supported MicroRNAs

描述（由申请人提供）：本项目的总体目标是了解胆管癌中致癌信号对microRNA的调节改变，以及microRNA如何有助于细胞死亡抵抗。颠覆生存途径的方法已经证明，肿瘤细胞并没有被赋予坚不可摧的盔甲，而是相当脆弱，并依赖于持续的促生存机制。最近，一个基于RNA的基因调控层已经出现，它可以促进肿瘤细胞的存活，其中microRNA的作用非常突出。microRNA以细胞类型和刺激特异性的方式对蛋白质组进行微调，并且改变的microRNA表达是人类癌症的特征。Sonic Hedgehog是一种在胆管癌中重新激活的发育途径，可能在microRNA改变中发挥作用。研究Hedgehog信号传导的microRNA靶标的初步研究已经鉴定了三种microRNA，mir-106 b，mir-93和mir-25，它们依赖于Hedgehog的表达。这些微小RNA聚集在7号染色体上，统称为mir-106 b ~mir-25。使用计算分析和细胞培养模型的额外初步数据已经证实，mir-25通过抑制死亡受体-4（两种促凋亡TRAIL受体之一）以及促凋亡BH 3蛋白Bim的表达来保护细胞免于TRAIL诱导的凋亡。基于这些观察，该提议的中心假设是Hedgehog信号传导通过增加mir-106 b-mir-25的表达和关键凋亡信号传导多肽DR 4和Bim的相互沉默来保护免于TRAIL诱导的死亡。具体目的是检验以下假设：（1）Hedgehog信号传导，通过MCM 7宿主基因转录，刺激mir-106 b ~mir-25产生;和（2）mir-25、mir-93和mir-106 b通过直接靶向死亡受体-4和Bim mRNA来保护细胞免于凋亡。已经开发了先进的分子和细胞工具来操纵microRNA和Hedgehog信号传导，以解决这些在肝胆肿瘤中具有生物医学重要性的问题。本申请寻求推进关于胆管癌中细胞死亡信号传导的科学知识。因此，拟议实验的结果与通过了解促进肝癌细胞死亡抵抗的改变途径来改善健康有关。该提案的成功完成将提供所需的机制见解，以影响通过Hedgehog抑制迫使胆管癌细胞死亡的临床方法。 公共卫生相关性：在绝大多数情况下，肝脏中的胆管癌是无法治愈的，原因之一是这种癌症增强了对细胞死亡的防御信号;通常为健康的早期发育保留的信号。我们建议研究这些发育信号如何阻止细胞死亡，并最终击败肿瘤生存的线索。这些结果有助于理解为什么患病的肿瘤细胞不受细胞死亡的保护，并可能导致更有效的肝癌治疗。

DESCRIPTION (provided by applicant): The OVERALL OBJECTIVES of this project are to understand the altered regulation of microRNAs by oncogenic signals in cholangiocarcinoma, and how microRNAs contribute to cell death resistance. Approaches that subvert survival pathways have demonstrated that tumor cells are not endowed with impenetrable armor, but are rather fragile, and dependent on ongoing prosurvival mechanisms. Recently, an RNA-based layer of gene regulation has emerged that promotes tumor cell survival, with a prominent role for microRNAs. MicroRNAs act to fine tune the proteome in a cell-type- and stimulus-specific manner and altered microRNA expression is a feature of human cancers. Sonic Hedgehog, a developmental pathway reactivated in cholangiocarcinoma, may play a role in microRNA alterations. Preliminary studies investigating microRNA targets of Hedgehog signaling have identified three microRNAs, mir-106b, mir-93, and mir-25 that are dependent upon Hedgehog for expression. These microRNAs are clustered on chromosome 7 and collectively referred to as mir-106b~mir-25. Additional preliminary data using computational analysis and cell culture models has confirmed that mir-25 protects cells from TRAIL-induced apoptosis by repressing expression of Death Receptor-4 (one of two pro-apoptotic TRAIL receptors) as well as the pro-apoptotic BH3 protein Bim. Based on these observations, the CENTRAL HYPOTHESIS of this proposal is that Hedgehog signaling protects against TRAIL-induced death via increased expression of mir-106b~mir-25 and reciprocal silencing of key apoptotic signaling polypeptides, DR4 and Bim. The SPECIFIC AIMS are to test the hypotheses that: (1) Hedgehog signaling, by MCM7 host-gene transcription, stimulates mir-106b~mir-25 production; and (2) mir-25, mir-93, and mir-106b protect cells from apoptosis by directly targeting Death Receptor-4 and Bim mRNAs. Advanced molecular and cellular tools to manipulate microRNAs and Hedgehog signaling have been developed to address these questions of biomedical importance in hepatobiliary neoplasia. The current application seeks to advance the scientific knowledge regarding cell death signaling in cholangiocarcinoma. Thus, the results of the proposed experiments are related to improving health by understanding the altered pathways promoting cell death resistance in liver cancer. Successful completion of this proposal will provide needed mechanistic insight to influence clinical approaches to compel cholangiocarcinoma cell death through Hedgehog inhibition. PUBLIC HEALTH RELEVANCE: Cancer of the bile ducts in the liver is incurable in the vast majority of cases, and one reason is that this cancer has heightened defensive signals against cell death; signals usually reserved for healthy early development. We propose studies on how these developmental signals act to block cell death, and ultimately to defeat tumor survival cues. The results are relevant to understanding why diseased tumor cells are protected from cell death and may lead to more effective liver cancer treatment.