Unraveling the MALAT1 lncRNA-protein interaction networks that drive lung cancer metastasis

揭示驱动肺癌转移的 MALAT1 lncRNA-蛋白质相互作用网络

• 批准号: 10283482
• 负责人: Chase A Weidmann
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10283482
• 关键词: Adenocarcinoma; Automobile Driving; Behavior; Binding Proteins; Bioinformatics; Biological Process; Cancer Biology; Cancer cell line; Cell Culture Techniques; Cell Line; Cell Proliferation; Cells; Chemicals; Chromatin; Complex; Cytoplasmic Granules; DNA; Data; Development; Disease; Drug resistance; Epithelial Cells; Event; Fluorescent in Situ Hybridization; Foundations; Functional disorder; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Goals; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Immunofluorescence Microscopy; Laboratories; Link; Lung; Lung Adenocarcinoma; MALAT1 gene; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Mass Spectrum Analysis; Metastatic Neoplasm to the Lung; Metastatic to; Microscopy; Molecular; Molecular Conformation; Monitor; Mus; Neoplasm Metastasis; Normal Cell; Nuclear; Nucleic Acids; Organelles; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Prognosis; Prognostic Marker; Proteins; Proteomics; RNA; RNA Splicing; RNA-Binding Proteins; RNA-Protein Interaction; Regulator Genes; Regulatory Pathway; Research; Resolution; Ribonucleoproteins; Role; Route; Signal Pathway; Structure; Technology; Testing; Therapeutic; Transcript; Translations; Untranslated RNA; Validation; Vision; Work; airway epithelium; base; cancer cell; cancer risk; cancer therapy; cancer type; career; cell motility; experience; experimental study; follow-up; improved; insight; lung cancer cell; lung metastatic; metaplastic cell transformation; migration; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; programs; protein complex; protein expression; stoichiometry; therapeutic target; transcriptome; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Long non-coding RNAs (lncRNAs), through interactions with RNA-binding proteins, play critical roles in development and cellular homeostasis. Many lncRNAs are associated with cancer and disease, representing a largely untouched pool of potential therapeutic targets. Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a highly conserved lncRNA whose expression is associated with poor prognosis and drug- resistance and whose activity accelerates cell proliferation and increases metastatic potential in multiple cancer types. MALAT1 is ubiquitously expressed at high levels in many normal cell lines, and yet genetic deletion of the transcript has no negative effects on development or viability in mice. The mechanisms of MALAT1 in both normal and cancer cells are unclear, and a better understanding of these mechanisms would unlock the potential of MALAT1 as a therapeutic target in the treatment of cancer. My preliminary data show that while levels of MALAT1 transcript are lower in a lung adenocarcinoma cell line than in normal human lung airway epithelial cells, metastatic activity of the adenocarcinoma is dependent on MALAT1: therefore high expression of MALAT1 is not sufficient to promote metastasis. I observe no differences in MALAT1 RNA structure between cell lines, but levels of expression of many MALAT1-binding proteins increase significantly. These data strongly indicate that proteins present in lung cancer cells are imparting pro-metastatic activity onto MALAT1. My goal is to integrate cutting-edge sequencing and quantitative proteomics technologies with cell-based functional approaches to understand how RNA-protein interaction networks of MALAT1 promote metastatic activity in human lung cancers. In Aim 1, I will purify MALAT1 RNA-protein complexes (RNPs) from normal lung cells and lung cancer cells and mechanistically detail the RNA and protein components of metastases-specific RNA- protein interaction networks. I will express fragments of MALAT1 in lung cancer cell lines and identify sequences sufficient to assemble RNP complexes and reprogram metastatic behavior in cell culture. In Aim 2, I will employ RBPome-wide total and phospho-protemics, nucleic acid interactome capture, and fluorescent microscopy to understand the signaling pathways that assemble dysfunctional MALAT1 RNPs genome- and transcriptome- wide. Together, these experiments will identify the mechanisms by which MALAT1-protein interaction networks promote each step of metastasis in lung cancer. I expect that this work will improve our fundamental understanding of MALAT1, inform strategies to target MALAT1 therapeutically, and act as a framework for the characterization of pro-metastatic lncRNA-protein complexes throughout the transcriptome. Development of these proposed aims will lay the foundation for a successful career as a leader in a new field of cancer RBPomes.

项目摘要/摘要 通过与RNA结合蛋白的相互作用，长的非编码RNA（LNCRNA）在 发展和细胞稳态。许多LNCRNA与癌症和疾病有关，代表 潜在的治疗靶标很大程度上没有触及的池。转移相关的肺腺癌转录本 1（malat1）是一种高度保守的lncRNA，其表达与预后不良和药物有关 抗性并且其活性加速了细胞增殖并增加了多种癌症的转移潜力 类型。在许多正常细胞系中，Malat1在高水平上的普遍表达，但遗传缺失的遗传缺失 转录物对小鼠的发育或生存能力没有负面影响。 Malat1的机制 正常和癌细胞尚不清楚，对这些机制的更好理解将释放潜力 Malat1作为癌症治疗的治疗靶标。我的初步数据表明，虽然 在肺腺癌细胞系中，MALAT1转录本低于正常的人肺气道上皮 细胞，腺癌的转移活性取决于Malat1：因此，Malat1的高表达 不足以促进转移。我观察到细胞系之间的malat1 RNA结构没有差异， 但是许多MALAT1结合蛋白的表达水平显着增加。这些数据强烈指出 肺癌细胞中存在的蛋白质正在向MALAT1赋予促次转移活性。我的目标是 将最先进的测序和定量蛋白质组学技术与基于细胞的功能相结合 了解MALAT1的RNA-蛋白相互作用网络如何促进转移性活动的方法 人类肺癌。在AIM 1中，我将从正常肺细胞和 肺癌细胞，并机械详细详细介绍转移酶特异性RNA的RNA和蛋白质成分 蛋白质相互作用网络。我将在肺癌细胞系中表达MALAT1的片段并识别序列 足以在细胞培养中组装RNP复合物和重编程转移行为。在AIM 2中，我会雇用 全rbPome范围的总和磷酸 - 蛋白质，核酸相互作用组捕获以及荧光显微镜 了解组装功能失调的MALAT1 RNP基因组和转录组的信号通路 宽的。这些实验将共同确定malat1-蛋白质相互作用网络的机制 促进肺癌转移的每个步骤。我希望这项工作将改善我们的基本 了解Malat1，告知策略以治疗目标MALAT1，并作为框架 整个转录组中促lncRNA-蛋白质复合物的表征。发展 这些拟议的目标将为在新的癌症RBPOMES领域取得成功的职业生涯奠定基础。