Stress and proliferation states impact microRNA-mediated regulation in cancer

描述（由申请人提供）：microRNAs水平的变化和microRNAs过表达的影响已经与人类恶性肿瘤有关。然而，microrna控制细胞状态的机制以及这种控制在应激条件下以及在静止细胞和增殖细胞中是如何改变的尚未被研究。我们提供的初步数据表明，microrna介导的调控在细胞对应激的反应中是重要的，并建议在正常细胞和恶性细胞中研究这种关系。初步的生物信息学分析表明，许多作为microRNA调控靶点的mRNA也含有RNA结合蛋白的保守位点，这些蛋白已知在应激过程中控制mRNA的翻译和稳定性。被microRNAs和应激相关RNA结合蛋白靶向的mrna在应激过程中可以优先表达。我们建议使用细胞培养氨基酸稳定同位素标记（SILAC）来表征应激和非应激条件下蛋白质与Argonaute/microRNA复合物的差异结合。我们提出初步证据，在这两种条件下检测结合蛋白的差异。这些蛋白将分析其在细胞过程中的作用，如翻译调节、亚细胞靶向和mRNA稳定性。我们进一步建议通过选择性免疫沉淀来自Dicer阴性胚胎干细胞的Argonaute结合mrna来鉴定microRNA靶向的mrna的总集合，因此缺乏内源性microRNA，这些microRNA被单个microRNA转染。总之，这些实验应该揭示应激条件下microRNA调控的重要性，并可以确定可用于优先抑制/杀死发生应激相关肿瘤的肿瘤细胞的药物靶点。在肿瘤发生过程中，如果3' utr中的靶位点消失，microrna的基因调控也会发生变化。我们发现大量基因在增殖过程中以短的3' utr表达，而在静止细胞中以长3' utr表达。当使用阵列比较静息CD4- t细胞和受体刺激的CD4细胞的mRNA表达时，观察到这种变化。进一步的生物信息学分析表明，这种转变发生在大多数静止组织和增殖组织以及肿瘤组织和正常组织中。静止细胞中的长3' utr几乎可以肯定地介导增强的microRNA调控，因为它们包含保守的种子靶位。我们建议通过研究控制增殖依赖性转变的因素的性质，这种变化在microRNA控制恶性表型中的重要性，以及如何调节这种转变以诱导癌细胞中更多的microRNA调节来继续这一分析。公共卫生相关性：癌症在公共卫生方面的负担在人类痛苦和医疗保健费用方面都是显而易见的。拟议的研究将为更好地治疗癌症的新疗法提供基础，从而改善公众健康。

DESCRIPTION (provided by applicant): Changes in the levels of microRNAs and effects of over-expression of microRNAs have already been related to human malignancy. However, the mechanisms by which microRNAs control cellular states and how this control is altered under stress conditions and in stationary verses proliferating cells have not been investigated. We present preliminary data indicating that microRNA-mediated regulation is important during the cell's response to stress and propose to study this relationship in normal and malignant cells. Preliminary bioinformatic analysis suggests that many mRNAs that are targets of microRNA regulation also contain conserved sites for RNA binding proteins that are known to control translation and mRNA stability during stress. mRNAs targeted by both microRNAs and stress-related RNA binding proteins can be preferentially expressed during stress. We propose to characterize the differential binding of proteins to Argonaute/microRNA complexes under stress and non-stress conditions using Stable Isotope Labeling with Amino acids in Cell culture (SILAC). We present preliminary evidence detecting differences in bound proteins under these two conditions. These proteins will be analyzed for their roles in cellular processes such as translational regulation, subcellular targeting, and mRNA stability. We further propose to identify the total set of mRNAs targeted by microRNAs by selective immunoprecipitation of Argonaute bound mRNAs from Dicer- negative embryonic stem cells, thus deficient in endogenous microRNAs that have been transfected with a single microRNA. In total, these experiments should reveal the importance of microRNA- regulation during stress conditions and could identify drug targets that could be used to preferentially inhibit/kill tumor cells undergoing stress-associated tumorigenesis. Gene regulation by microRNAs could also change during tumorigenesis if the target sites in 3' UTRs disappear. We have discovered that a large number of genes is expressed with short 3' UTRs during proliferation and longer 3' UTRs in quiescent cells. This shift was observed when arrays were used to compare mRNA expression in resting CD4-T cells and receptor stimulated CD4 cells. Further bioinformatic analysis shows this shift occurs in most resting verses proliferating tissues and in tumor verses normal tissue. The long 3' UTRs in resting cells almost certainly mediate enhanced microRNA regulation because they contain conserved seed target sites. We proposed to continue this analysis by investigating the nature of the factors controlling the proliferation-dependent shift, the importance of this change in microRNA control of the malignant phenotype, and how this shift can be modulated to induce more microRNA regulation in cancer cells. PUBLIC HEALTH RELEVANCE: The burden of cancer in public health is apparent in both human suffering and the cost of healthcare. The proposed research will provide the basis for new therapeutics to better treat cancer and thus improve public health.