Post-transcriptional coordination by HuR and microRNAs during tumorigenesis

描述（由申请人提供）：本申请旨在研究人类乳腺癌进展的RNA调控基础。众所周知，基因表达异常是癌症的一个重要因素，但对于rna结合蛋白（rbp）在疾病发生和进展中的作用知之甚少。序列特异性rbp协调调节功能相关mrna的亚群，形成核糖核蛋白复合物（RNPs），在小分子药物激活细胞时进行重塑。本提案的总体目标是确定由RBP人抗原R （HuR）协调的瞬时细胞RNP复合物促进或抑制肿瘤发生的动力学。已知在许多癌症中，HuR的调节发生了改变。研究表明，它可以稳定编码已知细胞生长调节因子和原癌基因的mRNA靶标，研究表明，与癌细胞相比，正常细胞中的HuR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水平与癌变之间存在很强的相关性。此外，最近的研究提出了一种机制，即HuR能够与microrna竞争以稳定mRNA靶标。因此，HuR可能通过抑制典型的抑制性microrna来差异调节编码肿瘤发生相关因子的mrna的稳定性，从而导致癌症相关蛋白的协调合成和疾病进展的加快。为了验证这一假设，rnp免疫沉淀和高通量测序将被用于识别和绘制人类乳腺癌恶性病程中与HuR相关的mRNA亚群的重塑。本课程将量化从正常人类乳腺上皮细胞到完全恶性表型的逐步转变。类似的研究将同时进行，以鉴定与Argonaute2蛋白（microRNPs的主要RBP成分）相关的mRNA亚群，以比较microRNAs在同一时间过程中靶向的mRNA。此外，将结合完善的分子和生化技术，包括荧光素酶构建、敲除和拯救实验、免疫荧光和RNA亲和沉淀测定，具体确定HuR从microrna介导的抑制中拯救mRNA靶标的机制细节。这项工作将在基因表达的转录后协调水平上提供有关致癌的潜在生物学信息，从而更全面地了解复杂基因调控的许多层面。这些研究中产生的转录后特征可用于鉴定HuR的小分子效应。因此，这项研究的结果可能最终导致对抗导致恶性肿瘤晚期的调节变化的能力。

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.