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Post-transcriptional coordination by HuR and microRNAs during tumorigenesis

肿瘤发生过程中 HuR 和 microRNA 的转录后协调

基本信息

批准号：
9022448
负责人：
Laura Simone Bisogno
金额：
$ 3.39万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-03-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9022448
关键词：
3&apos Untranslated Regions Affect Affinity Antigens Biochemical Biological Assay Biology Breast Epithelial Cells Cell Culture Techniques Cells Complex Cyclins Data Disease Progression Dissociation Energy Transfer Epithelial Cells Event Gene Expression Gene Expression Profile Gene Expression Regulation Health Hereditary Breast Carcinoma High-Throughput Nucleotide Sequencing Human Immunofluorescence Immunologic Immunoprecipitation In Situ Hybridization In Vitro Knock-out Lead Link Luciferases Malignant Neoplasms Maps Mediating Messenger RNA Metabolism Methods MicroRNAs Modeling Molecular Mutation Normal Cell Nuclear Export Oncogenes Onset of illness Pathologic Processes Pattern Pharmaceutical Preparations Play Precipitation Prognostic Factor Proteins Proto-Oncogenes RNA RNA Splicing RNA-Binding Proteins Regulation Regulon Reporting Repression Ribonucleoproteins Role Running Signal Transduction Staging Techniques Technology Testing Time Transcript Transfer RNA Translations WNT Signaling Pathway Work breast tumorigenesis cancer cell cancer therapy carcinogenesis cell growth deep sequencing functional outcomes in vivo insight knock-down mRNA Instability mRNA Stability malignant breast neoplasm malignant phenotype malignant state new therapeutic target novel strategies protein expression research study response small molecule synergism tumor progression tumorigenesis

项目摘要

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.

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 调控的基础原理。已知异常基因表达是癌症的一个重要因素，但人们对 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，形成核糖核蛋白复合物 (RNP)，该复合物在小分子药物激活细胞过程中被重塑。该提案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发生改变。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，正常细胞中 HuR 的靶标是不同的。目前还没有与癌症相关的 HuR 突变的报道，但 HuR 水平与癌发生之间存在很强的相关性。此外，最近的研究提出了一种机制，HuR 能够与 microRNA 竞争以稳定 mRNA 靶点。因此，HuR 很可能通过抑制典型的抑制性 microRNA 来差异性地调节参与肿瘤发生的 mRNA 编码因子的稳定性，从而导致癌症相关蛋白的协调合成并促进疾病进展。为了检验这一假设，RNP 免疫沉淀和高通量测序将用于识别和绘制人类乳腺癌恶性肿瘤整个时间过程中与 HuR 相关的 mRNA 子集的重塑图谱。这个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。类似的研究将同时进行，以确定与 Argonaute2 蛋白（microRNP 的主要 RBP 成分）相关的 mRNA 子集，以比较同一时间过程中 microRNA 靶向的 mRNA。此外，结合成熟的分子和生化技术，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，将用于专门确定允许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转录后协调水平上提供有关致癌作用的基础生物学信息，从而更全面地了解复杂基因调控的多层。这些研究中产生的转录后特征可用于鉴定 HuR 的小分子效应子。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监管变化。