Characterizing aberrant alternative-splicing in breast cancer

乳腺癌中异常选择性剪接的特征

基本信息

批准号：
8690565
负责人：
Angela Garibaldi
金额：
$ 3.53万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-07 至 2016-09-06
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8690565
关键词：
Address Affect Alternative Splicing Apoptosis Automobile Driving Bioinformatics Breast Cancer Cell Cancer cell line Cancerous Cell Cycle Cell Line Cell physiology Chromatin Communities Data Decision Making Development Disease Progression Event Gene Family Genes Genetic Transcription Goals Kinetics Knowledge Link Messenger RNA Molecular Profiling Nature Normal Cell Output Polymerase Process Property Protein Isoforms Proteins Protocols documentation RNA Binding RNA Polymerase II RNA Splicing Regulation Site Techniques Testing Time Tissues Transcription Initiation Tumor Tissue Variant Western Blotting Work anticancer research cancer therapy deep sequencing exon skipping genome-wide high throughput technology inhibitor/antagonist mRNA Precursor malignant breast neoplasm new therapeutic target next generation protein function public health relevance research study success transcription factor tumor progression

项目摘要

DESCRIPTION (provided by applicant): Alternative pre-mRNA splicing in breast cancer is known to affect genes responsible for cell cycle and apoptosis through the creation of alternate mRNA isoforms. Several of these events have been characterized as breast cancer-specific and are linked with disease progression. Previous studies of alternative splicing in breast cancer have focused on characterizing specific genes and gene families and do not address the co- transcriptional nature of these events. Here, we propose experiments that will generate genome-wide profiles of breast cancer-specific alternative splicing that will address this knowledge gap. First, we will address the question which alternative mRNA isoforms are created by splicing. The first specific aim will identify and verify breast cancer-specific changes in alternative splicing. Expression profiles will be generated using computational analyses of high throughput data for breast cancer cell lines, tumor tissue, normal cell lines, and non-tumor tissue. Aberrations in alternative splicing will be verified using quantitative PCR and western blo analysis. Given the proposal that pre-mRNA splicing may occur co-transcriptionally, decisions dictating how genes are differentially spliced in breast cancer may be accomplished during this same window. In the second specific aim we will determine the extent to which alternative splicing decisions are made co-transcriptionally. The extent of splicing of nascent mRNA will be determined through high throughput sequencing. Chromatin- bound RNA will be isolated using a technique recently established in our lab. In order to investigate the time associated properties of splicing, we will use a reversible transcription initiation inhibitor to synchronize transcriptin in breast cancer cell lines. Nascent mRNAs will then be extracted at biologically relevant time points and subjected to high throughput sequencing. Computational analyses of these outputs will generate comprehensive snap shots of splicing events such as alternative splice site selection or exon skipping events. Understanding both the consequences and the mechanisms of alternative splicing is vital to developing the next generation of breast cancer therapies and screenings.

描述（由申请人提供）：已知乳腺癌中的替代前MRNA剪接会影响导致细胞周期和凋亡的基因，从而创建替代mRNA同工型。其中几个事件被认为是乳腺癌特异性的，并与疾病进展有关。先前关于乳腺癌替代剪接的研究重点是表征特定基因和基因家族，并且没有解决这些事件的转录性质。在这里，我们提出的实验将产生乳腺癌特异性替代剪接的全基因组曲线，以解决这一知识差距。首先，我们将解决哪些替代mRNA同工型通过剪接产生哪些替代mRNA。第一个特定目的将识别和验证乳腺癌特异性变化在替代剪接中。将使用用于乳腺癌细胞系，肿瘤组织，正常细胞系和非肿瘤组织的高吞吐量数据的计算分析来生成表达谱。替代剪接中的畸变将通过定量PCR和Western BLO分析来验证。鉴于可能会共同转录的MRNA剪接的建议，因此决定在同一窗口中可以完成基因差异化的决定。在第二个特定目的中，我们将确定替代剪接决策的共同转录的程度。新生mRNA的剪接程度将通过高通量测序确定。染色质结合的RNA将使用最近在我们的实验室中建立的技术分离。为了研究剪接的时间特性，我们将使用可逆的转录起始抑制剂在乳腺癌细胞系中同步转录蛋白。然后，新生的mRNA将在生物学相关的时间点上提取，并进行高吞吐量测序。这些输出的计算分析将产生剪接事件的全面快照，例如替代剪接站点选择或外显子跳过事件。了解替代剪接的后果和机制对于发展下一代乳腺癌疗法和筛查至关重要。