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

基因组规模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、发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，但也证实了肿瘤发生的遗传和细胞复杂性。基因沉默机制的识别，RNA干扰(RNAi)，最初在无脊椎动物，后来在哺乳动物细胞中，对于我们理解基因表达的调节和我们通过实验调节它的能力具有巨大的意义。这个项目的重点是假设RNAi诱导的分子和表型扰动将使人们深入了解癌症的生物学，并识别新的抗癌分子靶点。大多数研究利用RNAi机制进行功能丧失(LOF)分析的目的是对编码mRNAs的蛋白质进行基因特异性切割。利用基于内源RNA的基因沉默机制RNAi的技术已经迅速发展，用于剖析基因与功能的关系，并作为进一步进行分子靶标分析的手段。该项目的总体目标是加强基于RNAi的技术在癌症生物学研究中的应用。为了做到这一点，我们正专注于建立在分子和功能水平上可重复性评估RNAi效应的方案和分析方法，这些方法可用于适合于肿瘤发生研究的哺乳动物细胞系模型系统。我们正在应用优化和强大的方案，使用合成的siRNAs和其他RNAi效应器，如短发夹状RNAs(ShRNAs)，在细胞中诱导RNAi，并使用定量分析RNA的有效性。到目前为止，我们已经检查了对应于250多个人类基因的siRNA和对应于50个人类基因的150个shRNA克隆所介导的沉默(后一项研究最初在项目编号Z01 BC 010614下详细介绍，但这项工作现在已在该项目的工作范围内进行)。该项目对于协助和培训CCR研究人员应用核糖核酸干扰分析，以及建立针对成百上千个人类基因的核糖核酸干扰筛查方案(见Z01 BC 010615)，仍然至关重要。

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).