The Induction of Gene-specific RNAi Against Cancer-associated Genes

针对癌症相关基因的基因特异性 RNAi 的诱导

• 批准号: 8763150
• 负责人: Natasha Caplen
• 金额: $ 31.12万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763150
• 关键词: Affect; Bioinformatics; Biological Assay; Biological Models; Biomedical Research; CCR; Cancer Biology; Cell Line; Cell physiology; Cells; Childhood; Coupled; Dissection; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genome; Glioblastoma; Goals; Human; Intention; Invertebrates; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of prostate; Mammalian Cell; Mediating; Messenger RNA; Methods; Microarray Analysis; Molecular; Molecular Analysis; Molecular Profiling; Molecular Target; Pathway interactions; Predisposition; Process; Production; Proteins; Proteolysis; Protocols documentation; RNA; RNA Interference; Research Personnel; Role; System; Technology; Training; Work; base; cancer cell; cancer therapy; gene function; gene induction; insight; loss of function; novel; sarcoma; small hairpin RNA; tool; tumorigenesis

Genome scale molecular analysis has revolutionized the experimental process by which the genes and pathways that influence the initiation, progression, and treatment of cancer can be identified. This has opened up many exciting avenues of biomedical research, but it has also served to confirm the genetic and cellular complexity that underlies tumorigenesis. The identification of the gene silencing mechanism, RNA interference (RNAi), initially in invertebrates and later in mammalian cells, has had enormous implications for our understanding of the regulation of gene expression and our ability to modulate it experimentally. This project is focused on the hypothesis that molecular and phenotypic perturbations induced by RNAi will give insight into the biology of cancer and identify novel anti-cancer molecular targets. The intention of most studies exploiting the RNAi mechanism for loss of function (LOF) analysis is the gene-specific cleavage of protein encoding mRNAs. Technologies that exploit the endogenous RNA-based gene silencing mechanism, RNAi, have developed rapidly for the dissection of gene-function relationships and as a means of furthering molecular target analysis. The overall goal of this project is the enhanced application of RNAi-based technologies for the study of cancer biology. To do this we are focusing on establishing protocols and assays for the reproducible assessment of the effects of RNAi at a molecular and functional level that can be used in mammalian cell line model systems appropriate for the study of tumorigenesis. We are applying optimized and robust protocols for inducing RNAi in cells using synthetic siRNAs and other RNAi effectors, such as short hairpin RNAs (shRNAs) and the use of quantitative assays for analyzing the efficacy of RNA. To date we have examined the silencing mediated by RNAi effectors corresponding to several hundred human genes. Following the induction of RNAi we are applying methods that can assay multiple molecular and phenotypic end-points to assess the function of a protein within specific cancer related pathways and at a broader systems level. Methods we employ include multiplex mRNA assays that allow us to study the downstream effects of LOF on specific pathways and whole transcriptome expression profiling (microarray based analysis) that enable us to identify new roles for genes in an unbiased manner. To support this work we have begun to develop and assess bioinformatic tools that can help us to identify non-targeted, but sequence-specific, affects on gene expression following introduction an RNAi effector into a cell. Our molecular based assays are coupled with functional studies chosen on the basis of the putative or defined role of the specific protein under study. During FY13 have been involved in studies that are using RNAi based technologies to (1) study genes required for the survival of ovarian cancer cells, (2) investigate genes associated with susceptibility to prostate cancer, and (3) identify genes required for the survival of pediatric sarcoma. Finally, this project continues to be critical for assisting and training CCR Investigators in the application of RNAi analysis, and for the establishment of protocols for RNAi screens targeting thousands of human genes (see Z01 BC 010615).

基因组量表分子分析彻底改变了实验过程，可以鉴定出影响癌症开始，进展和治疗的基因和途径。这为生物医学研究开辟了许多令人兴奋的途径，但它也有助于确认造成肿瘤发生的遗传和细胞复杂性。基因沉默机制RNA干扰（RNAI）最初在无脊椎动物中，后来在哺乳动物细胞中的鉴定对我们对基因表达的调节的理解以及我们对其实验调节的能力具有巨大的意义。该项目的重点是以下假设：RNAi诱导的分子和表型扰动将使您深入了解癌症的生物学并鉴定新的抗癌分子靶标。利用RNAi机制丧失功能（LOF）分析的大多数研究的意图是编码mRNA的蛋白质的基因特异性切割。利用基于内源性RNA的基因沉默机制RNAi的技术已迅速开发用于解剖基因功能关系，并作为促进分子靶标分析的一种手段。该项目的总体目标是增强基于RNAi的技术在癌症生物学研究中的应用。为此，我们着重于建立方案和测定法，以可重复评估RNAi在分子和功能水平上的影响，该疗程可用于哺乳动物细胞系模型系统，适用于研究肿瘤发生。我们正在使用合成siRNA和其他RNAi效应子（例如短发夹RNA（SHRNA）（SHRNA）以及使用定量测定法来分析RNA的功效，我们正在使用合成的siRNA和其他RNAi效应子进行优化且可靠的方案，以诱导细胞中的RNAi。迄今为止，我们已经检查了由RNAi效应子介导的沉默，与数百个人类基因相对应。 RNAi诱导后，我们正在应用可以测定多个分子和表型终点的方法，以评估蛋白质在特定癌症相关途径和更广泛的系统水平上的功能。我们采用的方法包括多重mRNA分析，使我们能够研究LOF对特定途径和整个转录组表达分析（基于微阵列的分析）的下游影响，这使我们能够以公正的方式鉴定基因的新作用。为了支持这项工作，我们已经开始开发和评估生物信息学工具，这些工具可以帮助我们识别未靶向但序列特异性的，会影响基因表达，因为将RNAi效应子引入细胞后的基因表达。我们的基于分子的测定法与根据所研究的特定蛋白的推定或定义作用选择的功能研究结合使用。在2013财年期间，已经参与了（1）对卵巢癌细胞生存所需的研究基因的研究，（2）研究与前列腺癌易感性相关的基因，以及（3）鉴定儿科肉瘤生存所需的基因。最后，该项目对于协助和培训CCR研究人员的RNAi分析以及针对针对数千个人类基因的RNAi筛选方案的应用仍然至关重要（请参阅Z01 BC 010615）。