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，与乳腺癌相关的替代前mRNA剪接的许多变化。这些观察结果提出了这样一个问题：前信使核糖核酸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不是调控不当的结果，还是肿瘤中普遍的剪接缺陷的结果？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移除内含子的内在能力的变化引起的。在这里，我们将进行实验，以深入了解选择性剪接是否有助于乳腺癌的进展。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和生物信息学来表征乳腺癌特异性的选择性剪接。(1)使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全面列表。(2)利用实时定量聚合酶链式反应和定量蛋白质印迹分析，验证剪接调控因子在乳腺癌中差异表达的假说。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前mRNA剪接预测结合起来，以确定剪接调控网络的可能靶点。所采取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，以获得对剪接调控网络的重要新见解。拟议的实验利用最新的实验和分析方法来生成乳腺癌特有的基因表达快照，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替代的前mRNA剪接是否是乳腺癌生物学的重要贡献。将这些分子洞察力引入对不同分期乳腺癌患者组织的分析，将表明选择性剪接的变化在多大程度上有助于乳腺癌的进展，从而提供额外的筛查和结果预测的可能性。 与公共卫生相关：已经在多种乳腺癌基因中发现了前mRNA剪接变异，这表明广泛的异常和选择性剪接可能是乳腺癌的后果甚至原因。在这里，我们建议评估替代的前mRNA剪接在多大程度上促进乳腺癌的进展。当将这种分析应用于不同分期的乳腺癌患者时，所获得的分子洞察力的价值就会意识到，因为它们可能提供额外的筛查和结果预测的可能性。

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.