Post-transcriptional coordination by HuR and microRNAs during tumorigenesis

描述（由申请人提供）：本申请旨在研究对人类乳腺癌进展至关重要的RNA调控的基本原理。已知异常基因表达是癌症的重要因素，但对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作为RNA调节子协调调节功能相关mRNA的子集以形成核糖核蛋白复合物（RNP），其在用小分子药物活化细胞期间被重塑。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复合物的动力学，以促进或抑制肿瘤发生。已知HuR的调节在许多癌症中被改变。它已被证明可以稳定编码已知细胞生长调节剂和原癌基因的mRNA靶标，并且研究表明，与癌细胞相比，HuR的靶标在正常细胞中是不同的。目前还没有报道与癌症相关的HuR突变，但HuR水平与癌症发生之间存在很强的相关性。此外，最近的研究表明HuR能够与microRNA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增强。为了检验这一假设，RNP免疫沉淀，然后高通量测序将用于识别和映射在整个人类乳腺癌恶性肿瘤的时间过程中与HuR相关的mRNA亚群的重塑。该时间过程将量化从正常人乳腺上皮细胞到完全恶性表型的逐步转变。将平行进行类似的研究，以鉴定与Argonaute2蛋白（microRNP的主要RBP组分）相关的mRNA亚群，以比较相同时间过程中microRNA靶向的mRNA。此外，成熟的分子和生物化学技术的组合，包括荧光素酶构建体，敲除和拯救实验，免疫荧光和RNA亲和沉淀测定，将用于具体确定机制的细节，使HuR从microRNA介导的抑制中拯救mRNA靶点。这项工作将提供有关致癌的潜在生物学在基因表达的转录后协调水平的信息，从而更全面地了解复杂的基因调控的许多层面。在这些研究中产生的转录后签名可用于鉴定HuR的小分子效应物。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监管变化的能力。

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.