Post-transcriptional coordination by HuR and microRNAs during tumorigenesis

本申请旨在研究人类乳腺癌核心RNA调控的基本原理 进展基因表达异常是已知的癌症的一个重要因素，但知之甚少 关于RNA结合蛋白（RBP）在疾病发生和发展中的作用。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 在一些实施方案中，RNP是在用小分子药物活化细胞期间重塑的RNP复合物。整体 本提案的目的是确定由细胞内的RNP复合物协调的瞬时细胞RNP复合物的动力学。 RBP人抗原R（HuR）促进或抑制肿瘤发生。已知HuR的调节会改变 在许多癌症中。已显示其稳定编码已知细胞生长调节剂的mRNA靶标， 原癌基因，研究表明，HuR的目标是不同的，在正常细胞相比， 癌细胞目前还没有报道与癌症相关的HuR突变，但在肿瘤的发生和发展之间存在很强的相关性。 HuR水平和致癌作用。此外，最近的研究表明，HuR能够 与microRNA竞争以稳定mRNA靶标。因此，很可能HuR差异性调节 通过抑制典型的抑制性微小RNA， 导致癌症相关蛋白质的协调合成和疾病进展的增强。 为了检验这一假设，将使用RNP免疫沉淀，然后进行高通量测序， 鉴定和绘制人乳腺在整个时间过程中与HuR相关mRNA亚群的重塑 恶性肿瘤该时间过程将量化从正常人类乳腺癌的逐步转变。 上皮细胞转化为完全恶性表型。类似的研究将平行进行，以确定mRNA 与Argonaute2蛋白（microRNP的主要RBP组分）相关的子集，以比较mRNA 在相同的时间过程中被microRNA靶向。此外，一个完善的 分子和生物化学技术，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特异性地确定 机制细节，使HuR从microRNA介导的抑制中拯救mRNA靶点。这项工作 将在转录后水平上提供关于致癌的潜在生物学信息， 基因表达的协调，从而更全面地了解许多层次的 复杂的基因调控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能够 对抗导致恶性肿瘤晚期的调节变化。

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.