Dynamics of Gene and Isoform Regulation during EMT and tumor progression

描述（由申请人提供）：上皮-间充质转化（EMT）是一种复杂的细胞生物学程序，在癌细胞进展为高度恶性肿瘤期间起作用，赋予这些细胞许多与侵袭性肿瘤相关的属性，包括扩散至远处部位和接种转移性集落的能力。这 该程序由一系列多效性作用的主转录因子（EMT-TF）协调，这些转录因子组织基因表达中的复杂变化，导致大量上皮细胞蛋白质被与间充质细胞状态相关的蛋白质取代。然而，对侵袭性间充质状态表达所需的主要关键控制水平知之甚少：在EMT期间表达变化的许多mRNA的前体也经历选择性剪接（AS），其赋予所得成熟的加工mRNA改变的性质，包括稳定性、蛋白质编码信息和对microRNA介导的抑制的响应性的变化。目前对AS对EMT程序执行的影响的零碎见解使得不可能对这一关键的细胞生物学程序如何受到影响形成合理完整的理解。拟议的研究将开始列举了数百个AS事件发生在几个替代机制诱导EMT程序在培养细胞和活组织。这样做之后，将采用生物信息学算法来确定与所涉及的剪接位点相邻的序列。此后，使用大量已经表征的RNA结合、剪接调节蛋白的已知核苷酸识别特性，将通过这些算法对可能B在通过EMT期间发生的AS中观察到的大规模移位的剪接调节剂的身份进行预测。这种实验策略应该产生AS的关键调节者的身份，这些调节者在执行EMT程序中可能与EMT-TF在功能上一样重要。将进行旨在功能性测试这些AS因素的候选性的实验测试。这些测试将衡量这些因子的强制或阻断表达是否影响EMT程序的关键组分的执行，以及是否如预测的那样，AS因子表达的这种强制变化影响关键EMT相关蛋白质的产生，即，在恶性进展期间观察到的上皮细胞与间充质细胞表型的表达中起关键作用的蛋白质。这项工作也有可能确定新的生物标志物的EMT程序，适用于，例如，在各种上皮组织中的干细胞的检测。

DESCRIPTION (provided by applicant): The epithelial-mesenchymal transition (EMT) is a complex cell-biological program that operates during the progression of carcinoma cells to high-grade malignancy, conferring on these cells many of the attributes associated with aggressive tumors, including the ability to disseminate to distant sites and to seed metastatic colonies. This program is orchestrated by a series of pleiotropically acting master transcription factors (EMT- TFs) that organize the complex changes in gene expression causing the replacement of a large cohort of epithelial cell proteins with those associated with the mesenchymal cell state. A major, critical level of control required for expression of the aggressive mesenchymal state is poorly understood however: the precursors of many of the mRNAs whose expression changes during the EMT also undergo alternative splicing (AS) that confer on resulting mature, processed mRNAs altered properties, including changes in stability, protein-coding information, and responsiveness to microRNA-mediated inhibition. The current fragmentary insights into the effects of AS on the execution of the EMT program make it impossible to form a reasonably complete understanding of how this critical cell-biological program is effected. The proposed research will begin by enumerating the hundreds of AS events that occur in response to several alternative mechanisms of inducing an EMT program both in cultured cells and in a living tissue. Having done so, bioinformatics algorithms will be employed to determine the sequences adjacent to involved splice sites. Thereafter, using the known nucleotide-recognizing properties of the large array of already-characterized RNA- binding, splice-regulating proteins, predictions will be made by these algorithms about the identities of the splice-regulators that are likely to b responsible for the observed large-scale shifts in AS occurring during passage through an EMT. This experimental strategy should yield the identities of key regulators of AS that are likely to b as important functionally as the EMT-TFs in executing the EMT program. Experimental tests designed to functionally test the candidacies of these AS factors will be performed. These tests will gauge whether the forced or blocked expression of these factors affect execution of critical components of the EMT program, and whether, as predicted, such imposed changes in AS factor expression affect the production of key EMT-associated proteins, i.e., proteins that play key roles in the expression of the epithelial versus mesenchymal cell phenotypes observed during malignant progression. This work also has the potential to identify novel biomarkers of the EMT program that are applicable, for example, for the detections of stem cells in a variety of epithelial tissues.