A new technology to isolate RNP complexes of a polycistronic miRNA oncogene

分离多顺反子 miRNA 癌基因 RNP 复合物的新技术

基本信息

批准号：
8641681
负责人：
Lin He
金额：
$ 19.42万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8641681
关键词：
Affinity Animal Model Animals Area B-Cell Lymphomas B-Lymphocytes Binding Biochemical Biogenesis Biological Biological Assay Biological Markers Biological Process Burkitt Lymphoma Candidate Disease Gene Cell Extracts Cell physiology Cells Code Complex Development Engineering Functional RNA Gene Expression Regulation Genetic Human In Vitro Individual Investigation Lead Lesion Malignant - descriptor Malignant Neoplasms Messenger RNA Methodology MicroRNAs Modeling Molecular Nucleotides Oncogenes Oncogenic Physiological Premalignant Proteins RNA RNA Sequences Regulation Ribonucleoproteins Role System Technology Transcript Transgenes Tumor Suppressor Genes Tumor Suppressor Proteins anticancer research base cancer cell endonuclease gene function in vivo insight mouse model new technology novel novel diagnostics public health relevance research study response therapeutic target tumor tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Malignant transformation represents the phenotypic endpoint of successive genetic lesions that alter gene function and gene regulation in oncogene and tumor suppressor networks. While extensive investigations have characterized protein-coding gene functions in these molecular networks, the functional importance of non-coding RNAs (ncRNAs) is just beginning to be revealed. microRNAs (miRNAs) encode a class of small regulatory ncRNAs with great capacity for post-transcriptional gene regulation by targeting a broad range of mRNAs. Our previous studies identified an important miRNA oncogene, mir17-92, that promotes the development of B-cell lymphomas. mir17-92 encodes a polycistronic miRNA transcript that yields six individual miRNA components, which have distinct biological functions and differential gene regulation during tumorigenesis. This context-dependent differential regulation of polycistronic miRNA components could ultimately determine the oncogenic activity of mir17-92. Aberrant regulation of miRNA biogenesis, stability and function has been frequently observed in human cancer, yet the underlying molecular basis still remains unclear. This is largely due to the lack of an effective methodology to study cancer-related miRNA ribonucleoprotein (miRNP) complexes. Here, we propose to develop a powerful biochemical strategy to isolate and characterize specific miRNA ribonucleoprotein (miRNP) complexes with important functions during tumorigenesis. We engineered a bacterial endonuclease, Csy4, to achieve high affinity binding and inducible cleavage of a specific 16-nucleotide (nt) RNA sequence. Using this 16-nt RNA as a tag, we aim to isolate specific miRNP complexes both in vitro and in vivo. These proposed studies will allow us to identify important miRNP complex components that differentially regulate components of a polycistronic miRNA oncogene in cancer cells. More importantly, our technology can be easily adapted to study cancer related RNP complexes containing other ncRNA species. With the increasing number of functionally important ncRNAs in cancer, our proposed studies will bring fundamental insights into an area of cancer research that has been largely unexplored.

描述（由申请人提供）：恶性转化代表连续遗传损伤的表型终点，这些损伤改变癌基因和肿瘤抑制网络中的基因功能和基因调控。虽然广泛的研究已经表征了这些分子网络中蛋白质编码基因的功能，但非编码 RNA (ncRNA) 的功能重要性才刚刚开始被揭示。 microRNA (miRNA) 编码一类小型调控 ncRNA，通过靶向广泛的 mRNA，具有强大的转录后基因调控能力。我们之前的研究发现了一个重要的 miRNA 癌基因 mir17-92，它可以促进 B 细胞淋巴瘤的发展。 mir17-92 编码多顺反子 miRNA 转录物，产生六个单独的 miRNA 组件，这些组件在肿瘤发生过程中具有不同的生物学功能和差异基因调控。这种多顺反子 miRNA 成分的上下文依赖性差异调节最终可以决定 mir17-92 的致癌活性。在人类癌症中经常观察到 miRNA 生物发生、稳定性和功能的异常调节，但潜在的分子基础仍不清楚。这主要是由于缺乏有效的方法来研究癌症相关 miRNA 核糖核蛋白 (miRNP) 复合物。在这里，我们建议开发一种强大的生化策略来分离和表征在肿瘤发生过程中具有重要功能的特定 miRNA 核糖核蛋白 (miRNP) 复合物。我们设计了一种细菌核酸内切酶 Csy4，以实现特定 16 核苷酸 (nt) RNA 序列的高亲和力结合和诱导切割。使用这个 16-nt RNA 作为标签，我们的目标是在体外和体内分离特定的 miRNP 复合物。这些拟议的研究将使我们能够识别重要的 miRNP 复合体成分，这些成分能够差异调节癌细胞中多顺反子 miRNA 癌基因的成分。更重要的是，我们的技术可以轻松地用于研究含有其他 ncRNA 种类的癌症相关 RNP 复合物。随着癌症中功能重要的 ncRNA 数量的不断增加，我们提出的研究将为癌症研究的一个很大程度上尚未探索的领域带来基本见解。