Post-transcriptional coordination by HuR and microRNAs during tumorigenesis

描述(由申请者提供)：本申请旨在研究人类乳腺癌进展过程中RNA调控的基本原理。基因异常表达被认为是癌症的一个重要因素，但对RNA结合蛋白(RBPs)在疾病发生和发展中所起的作用知之甚少。序列特异性RBPs作为RNA调节子协调调节功能相关的mRNAs的亚群，形成核糖核蛋白复合体(RNPs)，在小分子药物激活细胞时，RNPs被重塑。该建议的总体目标是确定由RBP人类抗原R(HUR)协调的瞬时细胞RNP复合体的动力学，以促进或抑制肿瘤的发生。众所周知，在许多癌症中，HUR的调控会发生变化。它已被证明可以稳定编码已知细胞生长调节因子和原癌基因的mRNA靶点，研究表明，与癌细胞相比，Hur在正常细胞中的靶标不同。目前还没有关于HUR与癌症相关的突变的报道，但HUR水平与癌症的发生有很强的相关性。此外，最近的研究表明，HUR能够与microRNAs竞争以稳定mRNA靶标的机制。因此，HUR可能通过抑制典型的抑制性microRNAs来差异地调节编码参与肿瘤发生的因子的mRNAs的稳定性，从而导致癌症相关蛋白的协调合成和促进疾病的进展。为了验证这一假设，RNP免疫沉淀和高通量测序将被用来识别和绘制与HUR相关的mRNA亚群在整个人类乳腺癌恶性过程中的重塑。这一时间进程将量化从正常的人类乳腺上皮细胞到完全恶性表型的逐步转变。将同时进行类似的研究，以确定与ArgAerte2蛋白(microRNPs的一个主要RBP成分)相关的mRNA亚群，以比较同一时间过程中microRNAs靶向的mRNAs。此外，将结合成熟的分子和生化技术，包括荧光素酶构建、基因敲除和挽救实验、免疫荧光和RNA亲和沉淀分析，来具体确定使Hur能够将mRNA靶标从microRNA介导的抑制中拯救出来的机制细节。这项工作将在转录后基因表达的协调水平上提供关于癌症发生的潜在生物学信息，从而更全面地理解复杂的基因调控的多个层次。在这些研究中产生的转录后签名可能被用来识别HUR的小分子效应物。因此，这项研究的结果最终可能导致对抗导致晚期恶性肿瘤的调节变化的能力。

DESCRIPTION (provided by applicant): This application aims to investigate the fundamentals of RNA regulation central to human breast cancer progression. Aberrant gene expression is known to be an important factor in cancer, yet very little is known about the role RNA-binding proteins (RBPs) play in disease onset and progression. Sequence specific RBPs coordinately regulate subsets of functionally related mRNAs as RNA regulons to form ribonucleoprotein complexes (RNPs), which are remodeled during activation of cells with small molecule drugs. The overall objective of this proposal is to determine the dynamics of transient cellular RNP complexes coordinated by the RBP human antigen R (HuR) to promote or suppress tumorigenesis. Regulation by HuR is known to be altered in many cancers. It has been shown to stabilize mRNA targets encoding known cell growth regulators and proto-oncogenes, and studies have demonstrated that targets of HuR are different in normal cells compared to cancer cells. No cancer-linked mutations of HuR have been reported, yet there is a strong correlation between HuR levels and carcinogenesis. In addition, recent studies suggest a mechanism whereby HuR is able to compete with microRNAs to stabilize mRNA targets. It is therefore likely that HuR differentially regulates the stability of mRNAs encoding factors involved in tumorigenesis by repressing typically suppressive microRNAs, resulting in the coordinated synthesis of cancer-associated proteins and enhancement of disease progression. To test this hypothesis, RNP-Immunoprecipitation followed by high-throughput sequencing will be used to identify and map remodeling of mRNA subsets associated with HuR throughout a time course of human breast cancer malignancy. This time course will quantify the stepwise transition from a normal human mammary epithelial cell to the fully malignant phenotype. Similar studies will be conducted in parallel to identify mRNA subsets associated with Argonaute2 protein (a major RBP component of microRNPs), to compare mRNAs targeted by microRNAs during the same time course. Furthermore, a combination of well-established molecular and biochemical techniques, including luciferase constructs, knockout and rescue experiments, immunofluorescence and RNA affinity precipitation assays, will be used to specifically determine the mechanistic details that allow HuR to rescue mRNA targets from microRNA-mediated suppression. This work will provide information about the underlying biology of carcinogenesis at the level of posttranscriptional coordination of gene expression, resulting in a more comprehensive understanding of the many layers of complex gene regulation. The posttranscriptional signatures generated in these studies may be used to identify small molecule effectors of HuR. Therefore, the results from this study may ultimately lead to the ability to counter the regulatory changes that contribute to advanced stages of malignancy.