Therapeutic use of let-7 and miR-34 microRNAs for the prevention and treatment of

let-7 和 miR-34 microRNA 在预防和治疗以下疾病中的治疗用途

• 批准号: 8003034
• 负责人: Andrea L Kasinski
• 金额: $ 4.76万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003034
• 关键词: Address; Adenocarcinoma; Aerosols; Alleles; Animals; Antineoplastic Agents; Apoptotic; Biological Assay; Cancer Etiology; Cancer cell line; Caspase; Cell Culture Techniques; Cell Cycle; Cells; Cessation of life; Control Groups; Disease; Epithelial Cells; Family; Growth; Harvest; Human; Infection; KRAS2 gene; Knowledge; Lead; Lung; Malignant Neoplasms; Malignant neoplasm of lung; MicroRNAs; Mutation; Pathway interactions; Patients; Prevention; Replacement Therapy; Research Design; TP53 gene; Testing; Therapeutic; Therapeutic Uses; Time; Tumor Burden; Woman; Work; cancer cell; cancer therapy; cell growth; lung tumorigenesis; men; molecular pathology; mutant; mutant mouse model; novel; overexpression; prevent; public health relevance; recombinase; response; tumor; vector; vector control

DESCRIPTION (provided by applicant): Lung cancer represents the leading cause of cancers deaths in both men and women worldwide, and current therapies fail to treat this disease in the overwhelming majority of cases. The RAS and p53 pathways are two of the most frequently genetically modified pathways in lung cancers. Alterations in both result in loss of responsiveness to current therapies leading to decreased overall patient survival. Tumor- suppressive microRNA (miRNA) families, including let-7 and miR-34, regulating steps in each of these pathways are often deleted in lung cancer, and their reduced expression is correlated with lung cancer. Objective/Hypothesis: In this work we propose to test the hypothesis that miRNA replacement therapy using miRNAs targeting the RAS pathway with those activated downstream of p53 will represent a novel yet powerful therapeutic to suppress lung tumorigenesis. Specific Aims: (1) To test the hypothesis that modifying let-7 and miR-34 microRNA levels will alter tumor- promoting ability in cell culture. (1a) We will test the effect of overexpression of let-7, miR-34, and the combination in a panel of human lung cancer cell lines with mutations in KRAS, p53 or both. (1b) We will evaluate the ability of anti-miRs to let-7, miR-34, and the combination to transform normal lung epithelial cells. (2) To test the hypothesis that overexpression of let-7 and miR-34 can both prevent and treat an inducible K- ras; p53 mutant mouse model of advanced lung cancer leading to an overall reduction in tumor burden. Study Design: We will address the hypothesis that miRNA treatment can reduce cell growth, invasiveness, and metastatic potential of lung cancer cells with activated KRAS and mutant p53. Cells will be transfected with one of the following: control vector, let-7 expressing vector, mir-34 expressing vector, or let-7/mir-34 expressing vector. Transfected cells will be evaluated by growth curves, ability to form colonies, cell cycle analysis and apoptotic assays such as phoshtidylserine externalization, and caspase activation. In parallel, animals with advanced lung cancer will evaluated for their response to let-7, miR-34 or the combination. For prevention studies, miRNAs will be administered at the same time as Cre recombinase, which induces expression of an activated K-ras allele, and the mutant p53 gene. A separate group of animals will only get Ad-Cre to determine bulk tumor burden in the absence of miRNA treatment. Animals will be sacrificed 8 weeks later, their lungs harvested and therapeutic potential evaluated. In subsequent studies we will evaluate the responsiveness of preformed tumors to miRNAs. Tumor formation will precede treatment by 6 weeks; a time that has been shown to produce early invasive adenocarcinomas in these compound animals. Animals will be sacrificed at 4 weeks post aerosol delivery of lent-let-7, -mir-34, or -let-7/mir-34 and the amount of proliferation in the lungs will be compared to a control group of animals sacrificed at the time of miRNA infection. PUBLIC HEALTH RELEVANCE: Our proposed work on let-7 and miR-34 families of microRNAs has the potential to provide a better understanding of the molecular pathology of lung cancer, knowledge that could lead to significant improvements in lung cancer's treatment and ultimate cure rates in the long term. While this is an emerging field, the benefit to our understanding of miRNAs in cancer will be enormous if we can harness these natural growth repressors as anti-cancer agents

描述（由申请人提供）：肺癌是全球男性和女性癌症死亡的主要原因，目前的治疗方法无法治疗绝大多数病例。RAS和p53通路是肺癌中最常见的两种遗传修饰通路。两者的改变导致对当前治疗的反应性丧失，从而导致患者总体生存期降低。肿瘤抑制性微小RNA（miRNA）家族，包括let-7和miR-34，这些途径中的每一个中的调节步骤通常在肺癌中缺失，并且它们的表达降低与肺癌相关。目的/假设：在这项工作中，我们提出了一个假设，即使用靶向RAS通路的miRNA与p53下游激活的miRNA替代疗法将代表一种新的但强大的治疗，以抑制肺肿瘤的发生。具体目的：（1）检验修饰let-7和miR-34 microRNA水平将改变细胞培养物中的肿瘤促进能力的假设。(1a)我们将在一组KRAS、p53或两者突变的人肺癌细胞系中测试let-7、miR-34的过表达及其组合的效果。(1b)我们将评估抗miR对let-7、miR-34和其组合转化正常肺上皮细胞的能力。(2)为了验证let-7和miR-34的过表达可以预防和治疗晚期肺癌的诱导型K-ras; p53突变小鼠模型，从而导致肿瘤负荷的总体降低的假设。研究设计：我们将提出这样的假设，即miRNA治疗可以降低具有激活的KRAS和突变型p53的肺癌细胞的细胞生长、侵袭力和转移潜力。细胞将用以下之一转染：对照载体、let-7表达载体、mir-34表达载体或let-7/mir-34表达载体。将通过生长曲线、形成集落的能力、细胞周期分析和凋亡测定如磷脂酰丝氨酸外化和半胱天冬酶活化来评价转染的细胞。 平行地，将评估患有晚期肺癌的动物对let-7、miR-34或组合的响应。对于预防研究，miRNA将与Cre重组酶同时施用，Cre重组酶诱导活化的K-ras等位基因和突变型p53基因的表达。单独的一组动物将仅获得Ad-Cre，以在不存在miRNA治疗的情况下确定整体肿瘤负荷。8周后处死动物，收获其肺并评价治疗潜力。 在随后的研究中，我们将评估预先形成的肿瘤对miRNA的反应性。肿瘤形成将在治疗前6周;这一时间已显示在这些化合物动物中产生早期浸润性腺癌。在气雾剂递送lent-let-7、-mir-34或-let-7/mir-34后4周处死动物，并将肺中的增殖量与在miRNA感染时处死的动物的对照组进行比较。 公共卫生相关性：我们提出的let-7和miR-34 microRNA家族的工作有可能更好地了解肺癌的分子病理学，这些知识可能导致肺癌治疗和最终治愈率的显着改善。虽然这是一个新兴的领域，但如果我们能够利用这些天然生长抑制因子作为抗癌剂，那么我们对癌症中miRNA的理解将是巨大的