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

基因组尺度的分子分析已经彻底改变了实验过程，通过它可以识别影响癌症发生、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，但它也证实了肿瘤发生背后的遗传和细胞复杂性。基因沉默机制，RNA干扰（RNAi）的鉴定，最初是在无脊椎动物中，后来在哺乳动物细胞中，对我们对基因表达调控的理解和我们实验调节它的能力有着巨大的影响。该项目的重点是假设由RNAi诱导的分子和表型扰动将深入了解癌症生物学并确定新的抗癌分子靶点。利用RNAi机制进行功能丧失（LOF）分析的大多数研究的目的是编码mrna的蛋白质的基因特异性切割。利用内源性rna为基础的基因沉默机制的技术，RNAi，已迅速发展，用于解剖基因-功能关系和进一步分子靶标分析的手段。该项目的总体目标是增强基于rna的技术在癌症生物学研究中的应用。为了做到这一点，我们专注于建立RNAi在分子和功能水平上的可重复评估的方案和分析，可用于适合研究肿瘤发生的哺乳动物细胞系模型系统。我们正在应用优化和稳健的方案，使用合成sirna和其他RNAi效应物（如短发夹RNA (shRNAs)）在细胞中诱导RNAi，并使用定量分析RNA的功效。到目前为止，我们已经研究了250多个人类基因对应的sirna介导的沉默和50个人类基因对应的150个shRNA克隆（后一项研究最初在项目编号Z01 BC 010614下详细介绍，但这项工作现在已经在该项目中进行了调整）。该项目对于协助和培训CCR研究人员应用RNAi分析以及建立针对数百个人类基因的RNAi筛选方案仍然至关重要（见Z01 BC 010615）。

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 siRNAs corresponding to over ≈250 human genes and ≈150 shRNA clones corresponding to ≈50 human genes (this latter study was originally detailed under project number Z01 BC 010614 but this work has now been aligned within this project effort). 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 hundreds of human genes (see Z01 BC 010615).