The role of long noncoding RNA CRNDE in normal physiology and cancer

项目摘要 越来越多的证据表明，长链非编码RNA（lncRNA），一类不同的非蛋白质- 长度>200个核苷酸的编码转录物在癌症的发生和发展中起重要作用。 已经提出lncRNA调节所有癌症标志，但是，在绝大多数情况下，它们的表达与肿瘤的发生有关。 作用的分子机制仍然知之甚少。这种知识差距是一个主要障碍 实现lncRNA作为癌症和其他疾病治疗靶点的潜力。因为在许多 在人类恶性肿瘤中，lncRNA在肾细胞癌（RCC）中经常失调。RCC是最 肾癌是一种常见的肾癌，也是最致命的恶性泌尿系统肿瘤，每年约有70，000例新发 美国每年确诊的病例数。迄今为止，大多数RCC（和其他癌症）的基因组研究 一直专注于识别与疾病相关的蛋白质编码基因的改变。我们理解 肾癌中lncRNA调控的分子途径，以及这些转录本在这一过程中的功能作用。 恶性肿瘤，仍然有限。我们从RCC患者中挖掘RNA-seq数据，以确定一组805种lncRNA， 通常在这种肿瘤中过度表达。我们生成了定制的CRISPR干扰（CRISPRi） 靶向这些lncRNA的文库，并在多个RCC细胞系中进行筛选，以鉴定 对RCC细胞增殖至关重要。这些实验表明，lncRNA结肠直肠肿瘤 差异表达（CRNDE）是所有测试的RCC细胞系的生长所需要的。尽管这种lncRNA 已被证明是过度表达，并与肾癌和其他类型的肿瘤患者的生存率低有关。 癌症，其分子功能仍不清楚。我们确定了CRNDE的一个关键区域， RCC细胞增殖，我们鉴定了与该序列相互作用的蛋白质。我们还创造了新的 在小鼠中进行基因工程改造的等位基因，使关键基因的组成性或条件性缺失成为可能。 序列的在这项提案中，我们将利用我们对这种lncRNA的新理解，以及新的资源， 为了剖析CRNDE的分子功能，并确定其在正常人中的作用， 生理学和体内RCC发病机制。这些实验将利用我们广泛的 经验，以及我们的合作者，在评估非编码RNA功能和RCC生物学使用 细胞和动物模型。成功完成拟议的研究将解决两个主要知识 RNA生物学和癌症生物学领域的差距：1）我们对分子生物学的理解有限， lncRNA的作用机制;以及2）这些机制如何被癌细胞吸收以促进肿瘤细胞的生长。 肿瘤生长，特别是在RCC中。我们预计，这些研究揭示的原则将广泛地 适用于我们对癌细胞中其他lncRNA作用的理解，并可能为 开发针对CRNDE或其控制的途径的治疗方法。

PROJECT SUMMARY A growing body of evidence indicates that long noncoding RNAs (lncRNAs), a diverse class of non-protein- coding transcripts >200 nucleotides in length, play important roles in the initiation and progression of cancer. LncRNAs have been proposed to regulate all cancer hallmarks, but, in the vast majority of cases, their molecular mechanisms of action remain poorly understood. This knowledge gap is a major impediment towards realizing the potential of lncRNAs as therapeutic targets in cancer and other diseases. As in many human malignancies, lncRNAs are frequently dysregulated in renal cell carcinoma (RCC). RCC is the most common type of kidney cancer and the most lethal malignant urological tumor, with approximately 70,000 new cases diagnosed annually in the United States. To date, most genomic studies of RCC (and other cancers) have focused on identifying disease-associated alterations of protein-coding genes. Our understanding of the molecular pathways regulated by lncRNAs in RCC, and the functional roles of these transcripts in this malignancy, remains limited. We mined RNA-seq data from RCC patients to identify a set of 805 lncRNAs that are commonly overexpressed in this tumor type. We generated a custom CRISPR interference (CRISPRi) library targeting these lncRNAs and performed screens in multiple RCC cell lines to identify lncRNAs that are essential for RCC cell proliferation. These experiments revealed that the lncRNA Colorectal neoplasia differentially expressed (CRNDE) is required for growth of all tested RCC cell lines. Although this lncRNA has been shown to be overexpressed and is associated with poor patient survival in RCC and other types of cancer, its molecular function remains unclear. We identified a critical region of CRNDE that is necessary for RCC cell proliferation and we identified proteins that interact with this sequence. We also generated novel genetically-engineered alleles in mice that enable constitutional or conditional deletion of critical Crnde sequences. In this proposal, we will leverage our new understanding of this lncRNA, and the novel resources we have generated, in order to dissect the molecular function of CRNDE and define its role in normal physiology and in RCC pathogenesis in vivo. These experiments will take advantage of our extensive experience, and that of our collaborators, in evaluating noncoding RNA functions and RCC biology using cellular and animal models. Successful completion of the proposed research will address two major knowledge gaps in the fields of RNA biology and cancer biology: 1) our limited understanding of the molecular mechanisms-of-action of lncRNAs; and 2) how these mechanisms are co-opted by cancer cells to promote tumor growth, particularly in RCC. We anticipate that the principles revealed by these studies will be broadly applicable to our understanding of the roles of other lncRNAs in cancer cells and may set the stage for developing therapeutics that target CRNDE or the pathways it controls.