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: 8316836
• 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/8316836
• 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. PUBLIC HEALTH RELEVANCE: The purpose of this project is to improve our understanding of the biology of pancreatic cancer. The proposed experiments will describe previously unexplored aspects of pancreatic cancer's initiation and progression in the body. A greater understanding of these mechanisms will contribute to the development of improved therapies for patients with this disease.

描述（由申请人提供）：胰腺导管腺癌（PDAC）是美国与癌症相关死亡的第四个主要原因，五年生存率少于5％，诊断6个月后的中位生存期。这些统计数据反映出缺乏有效的早期检测以及缺乏针对全身性疾病的有效治疗方法。在建立更有针对性和有效的疗法之前，需要对PDAC启动和进展的潜在生物学有更多的了解。拟议的工作将探讨PDAC的驾驶突变（突变激活的Krasg12d）与miR-17〜92 microRNA簇之间的潜在关系，特别解决了Kras增殖，亲卵形和肿瘤型胰腺和胰胰中性表型中MiR-17〜92的需求。为了确定KRASG12D诱导的细胞表型是否需要miR-17〜92 miRNA，将分离原代导管性上皮细胞（PDEC），并且将确定miR-17〜92缺失对KRASG12D诱导的增殖的影响，并确定在存在的杀菌性刺激刺激刺激刺激的情况下。 miRNA簇的成分将被外源表达，以评估特定miRNA对这些表型的潜在拯救。将在内源表达Krasg12d的动物中评估miR-17〜92簇对体内PDAC前体病变形成的缺失对PDAC前体病变形成的影响，并在胰腺中特异性删除了miR-17〜92簇。动物将在4、8和12个月大的年龄和胰腺的组织学部分处死，检查了前体病变的存在，数量和等级。使用小鼠模型，该模型还结合了TP53肿瘤抑制基因的杂合缺失，将确定miR-17〜92缺失对PDAC发育和进展的影响。这些研究是特定目标1的重点。本研究还将确定miR-17〜92簇和激活的KRAS之间的相互作用机制。通过使用表达KRASG12D的PDEC的Messenger RNA微阵列的多个目标预测算法和数据，将确定在PDEC中表达的miR-17〜92群集的可能目标。表达水平将在miR-17〜92野生型和NULL PDEC中通过蛋白质印迹评估，并且将通过将靶标3'UTRS融合到荧光素酶并测量有或没有miRNA簇的细胞中的发光来确认miRNA介导的抑制作用。 KRAS的下游途径活性将在群集无PDEC中测量，以解决17〜92是否通过调节特定的下游效应器途径调节KRASG12D诱导的表型。这些研究是特定目的2的重点。共同提出的研究将揭示miR-17〜92 miRNA簇在胰腺癌的启动和进展中的作用，从而为开发新型治疗方法奠定了基础。 公共卫生相关性：该项目的目的是提高我们对胰腺癌生物学的理解。提出的实验将描述胰腺癌开始和体内进展的先前未开发的方面。对这些机制的更多了解将有助于改善该疾病患者的疗法。