Roles of MiR-17-92 Cluster MicroRNAs in K-Ras-Induced Pancreatic Tumorigenesis

MiR-17-92 簇 MicroRNA 在 K-Ras 诱导的胰腺肿瘤发生中的作用

• 批准号: 8465753
• 负责人: Brian Joseph Quattrochi
• 金额: $ 2.87万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8465753
• 关键词: Address; Adenocarcinoma Cell; Affect; Age-Months; Algorithms; Animals; Apoptotic; Automobile Driving; Biology; Cancer Etiology; Carcinoma; Cell Line; Cell Proliferation; Cell Survival; Cells; Cessation of life; Data; Development; Diagnosis; Disease; Ductal Epithelial Cell; Early Diagnosis; Epithelial Cells; Foundations; Genes; Histologic; In Vitro; KRAS2 gene; Knowledge; Lesion; Light; Luciferases; MEKs; Malignant neoplasm of pancreas; Measures; Mediating; Messenger RNA; MicroRNAs; Molecular; Mutation; Oncogenic; Pancreas; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patients; Phenotype; Play; Property; Proteins; Proto-Oncogene Proteins c-akt; Regulation; Research; Role; Signal Pathway; Signal Transduction; Staging; Stimulus; Survival Rate; Systemic disease; TP53 gene; Therapeutic; Time; Tissues; Tumor Suppressor Genes; United States; Untranslated Regions; Western Blotting; Work; base; cancer initiation; effective therapy; improved; in vivo; luminescence; mouse model; novel therapeutic intervention; pancreatic tumorigenesis; research study; statistics; tumor; tumor progression; tumorigenesis; tumorigenic

DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with a five-year survival rate of less than 5%, and a median survival after diagnosis of 6 months. These statistics reflect the lack of effective early detection and the absence of effective treatments for systemic disease. Greater knowledge of the underlying biology of PDAC initiation and progression is required before more targeted and effective therapies can be developed. The proposed work will explore the potential relationship between the driving mutation of PDAC (mutationally activated KrasG12D) and the miR-17~92 cluster of microRNAs, specifically addressing the requirement for miR-17~92 in Kras' proliferative, pro-survival and tumorigenic phenotypes within the pancreas and the molecular mechanism behind this relationship. To determine whether miR-17~92 miRNAs are required for KrasG12D-induced cellular phenotypes, primary ductal epithelial cells (PDECs) will be isolated and the effect of miR-17~92 deletion on KrasG12D-induced proliferation and survival in the presence of apoptotic stimuli will be determined. Components of the miRNA cluster will be expressed exogenously to assess potential rescue of these phenotypes by specific miRNAs. The effect of deletion of the miR-17~92 cluster on PDAC precursor lesion formation in vivo will be assessed in animals that endogenously express KrasG12D and have deleted the miR-17~92 cluster specifically in the pancreas. Animals will be sacrificed at 4, 8, and 12 months of age and histologic sections of the pancreas examined for the presence, quantity and grade of precursor lesions. Using a mouse model that additionally incorporates heterozygous deletion of the TP53 tumor suppressor gene, the impact of miR-17~92 deletion on PDAC development and progression will be ascertained. These studies are the focus of Specific Aim 1. This study will also identify the mechanism of interaction between the miR-17~92 cluster and activated Kras. Through the use of multiple target prediction algorithms and data from a messenger RNA microarray profile of PDECs expressing KrasG12D, probable targets of the miR-17~92 cluster that are expressed in PDECs will be identified. Expression levels will be evaluated in miR-17~92 wild type and null PDECs by Western blot, and miRNA-mediated suppression will be confirmed by fusing target 3'UTRs to luciferase and measuring luminescence in cells with and without the miRNA cluster. Downstream pathway activity of Kras will be measured in cluster-null PDECs to address whether 17~92 modulates KrasG12D-induced phenotypes through the regulation of specific downstream effector pathways. These studies are the focus of Specific Aim 2. Together, the studies proposed in this application will shed light on the role of the miR-17~92 miRNA cluster in pancreatic cancer initiation and progression, potentially laying the foundation for the development of novel therapeutic approaches.

描述（由申请人提供）：胰腺导管腺癌（Pancreatic ductal adencarcinoma， PDAC）是美国第四大癌症相关死亡原因，5年生存率低于5%，诊断后中位生存期为6个月。这些统计数据反映了对系统性疾病缺乏有效的早期发现和有效的治疗。在开发更有针对性和更有效的治疗方法之前，需要对PDAC发生和进展的潜在生物学有更多的了解。本研究将探索PDAC（突变激活的KrasG12D）驱动突变与miR-17~92 microrna簇之间的潜在关系，特别是解决胰腺中Kras增殖、促生存和致瘤表型对miR-17~92的需求，以及这种关系背后的分子机制。为了确定krasg12d诱导的细胞表型是否需要miR-17~92 mirna，将分离原代导管上皮细胞（PDECs），并确定miR-17~92缺失对凋亡刺激下krasg12d诱导的增殖和存活的影响。miRNA集群的组成部分将被外源表达，以评估特定miRNA对这些表型的潜在拯救。在内源性表达KrasG12D并在胰腺特异性删除miR-17~92簇的动物中，将评估miR-17~92簇缺失对体内PDAC前体病变形成的影响。动物将在4、8和12个月大时被处死，胰腺的组织学切片检查前驱病变的存在、数量和等级。通过小鼠模型添加杂合缺失TP53肿瘤抑制基因，将确定miR-17~92缺失对PDAC发生和进展的影响。这些研究是Specific Aim 1的重点。本研究还将确定miR-17~92簇与活化的Kras之间相互作用的机制。通过使用多种靶标预测算法和来自表达KrasG12D的PDECs的信使RNA微阵列谱的数据，将确定PDECs中表达的miR-17~92簇的可能靶标。通过Western blot评估miR-17~92野生型和空型PDECs的表达水平，并通过将目标3'UTRs融合到荧光素酶并测量有和没有miRNA簇的细胞的发光来证实miRNA介导的抑制。Kras的下游通路活性将在无簇的PDECs中进行测量，以确定17~92是否通过调节特定的下游效应通路来调节krasg12d诱导的表型。这些研究是Specific Aim 2的重点。总之，本应用中提出的研究将阐明miR-17~92 miRNA簇在胰腺癌发生和进展中的作用，可能为开发新的治疗方法奠定基础。