The role of alternative pre-mRNA splicing in breast cancer progression

描述（由申请人提供）：最近的研究证明，许多替代前mrna剪接的变化与乳腺癌有关。这些观察结果提出了一个问题，即mrna前剪接的变化是否会导致乳腺癌？另一个悬而未决的问题是，乳腺癌特异性剪接事件是错误调控的结果，还是肿瘤中普遍剪接缺陷的结果？我们已经证明，与乳腺癌相关的选择性剪接事件不是由剪接体去除内含子的内在能力的变化引起的。在这里，我们将开展实验以深入了解选择性剪接是否有助于乳腺癌进展的问题。在这个应用中，我们将表征前mrna剪接网络，以测试剪接调节因子的异常表达触发乳腺癌特异性选择性剪接的假设。我们将结合实验分析与生物信息学表征乳腺癌特异性选择性剪接。(1)利用高通量测序方法和EST数据库的计算分析，我们将生成并验证乳腺癌特异性选择性剪接事件的综合列表。(2)采用real-time PCR和定量western blot分析，验证剪接调控因子在乳腺癌中存在差异表达的假设。我们将把这些表达谱与乳腺癌特异性选择性剪接事件的计算分析和mrna前剪接预测相结合，以确定剪接调节网络的可能靶点。所采用的方法将高通量数据生成与生物信息学和经典分子生物学方法相结合，以获得对剪接调节网络的重要新见解。拟议的实验利用最新的实验和分析方法来生成乳腺癌独特的基因表达快照，从而产生对乳腺癌研究界有用的丰富数据。所描述的实验将证明是否替代前mrna剪接是乳腺癌生物学的重要贡献者。将这些分子见解引入到不同阶段分组的乳腺癌患者的组织分析中，将表明选择性剪接的变化在多大程度上促进了乳腺癌的进展，从而提供额外的筛选和结果预测可能性。

DESCRIPTION (provided by applicant): Recent work has documented that many changes in alternative pre-mRNA splicing associate with breast cancer. These observations raise the question whether changes in pre-mRNA splicing contribute to breast cancer? Another unanswered question is whether breast cancer-specific splicing events are the result of mis-regulations or whether they are the consequence of a general splicing defect in tumors? We have demonstrated that alternative splicing events associated with breast cancer are not caused by changes in the intrinsic ability of the spliceosome to remove introns. Here, we will carry out experiments to gain insights into the question whether alternative splicing contributes to breast cancer progression. In this application, we will characterize pre-mRNA splicing networks to test the hypothesis that aberrant expression of splicing regulators trigger breast cancer-specific alternative splicing. We will combine experimental analyses with bioinformatics to characterize breast cancer-specific alternative splicing. (1) Using high throughput sequencing approaches and computational analyses of EST databases we will generate and validate a comprehensive list of breast cancer-specific alternative splicing events. (2) Using real-time PCR and quantitative western blot analysis, we will test the hypothesis that splicing regulators are differentially expressed in breast cancer. We will combine these expression profiles with computational analyses of breast cancer-specific alternative splicing events and pre-mRNA splicing predictions to determine likely targets of splicing regulatory networks. The approaches taken integrate high-throughput data generation with bioinformatics and classical molecular biology methods to gain important new insights into splicing regulatory networks. The proposed experiments take advantage of the most up-to-date experimental and analytical approaches to generate a snapshot of gene expression unique to breast cancer, thus producing a wealth of data useful to the breast cancer research community. The described experiments will demonstrate whether alternative pre-mRNA splicing is a significant contributor to breast cancer biology. Introducing these molecular insights to analyses of tissues obtained from breast cancer patients of various stage groupings will indicate to what degree changes in alternative splicing contribute to breast cancer progression, thus, providing additional screening and outcome prediction possibilities. PUBLIC HEALTH RELEVANCE: Pre-mRNA splice variants have been identified for a large variety of breast cancer genes, suggesting that widespread aberrant and alternative splicing may be a consequence or even a cause of breast cancer. Here, we propose to evaluate to what degree alternative pre-mRNA splicing contributes to breast cancer progression. The value of the molecular insights gained is realized when applying such analyses to breast cancer patients of various stage groupings, as they are likely to provide additional screening and outcome prediction possibilities.