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过度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压力条件下和静止状态下与增殖细胞相比是如何改变的，还没有被研究过。我们提供的初步数据表明，microRNA介导的调节在细胞对应激的反应中是重要的，并建议在正常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调控目标的mRNAs也包含RNA结合蛋白的保守位点，这些蛋白已知在应激过程中控制翻译和mRNA的稳定性。由microRNAs和应激相关RNA结合蛋白靶向的mRNAs在应激过程中可以优先表达。我们建议使用细胞培养中氨基酸的稳定同位素标记(SILAC)来表征应激和非应激条件下蛋白质与ArgAerte/microRNA复合体的差异结合。我们提出了在这两种情况下检测结合蛋白差异的初步证据。这些蛋白质将被分析它们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从DICER阴性胚胎干细胞中分离ArgAerte结合的mRNAs来鉴定microRNAs靶向的全部mRNAs，从而缺乏已转染单个microRNA的内源性microRNAs。总而言之，这些实验应该揭示在应激条件下微RNA调控的重要性，并可以确定可以用于优先抑制/杀死经历应激相关肿瘤形成的肿瘤细胞的药物靶点。如果3‘UTRs中的靶点消失，在肿瘤发生过程中，microRNAs对基因的调控也会发生变化。我们发现，大量的基因在增殖过程中表达短的3‘UTRs，在静止的细胞中表达较长的3’UTRs。当阵列被用来比较静止的CD4-T细胞和受体刺激的CD4细胞中的mRNA表达时，这种变化被观察到。进一步的生物信息学分析表明，这种转变发生在大多数静息和增殖组织中，以及肿瘤和正常组织中。静息细胞中的长3‘UTRs几乎肯定地介导了增强的microRNA调节，因为它们含有保守的种子靶点。我们建议继续这一分析，调查控制增殖依赖转变的因素的性质，这种改变在恶性表型的microRNA控制中的重要性，以及如何调节这种转变以在癌细胞中诱导更多的microRNA调节。公共卫生相关性：癌症在公共卫生中的负担在人类痛苦和医疗保健成本中都是显而易见的。这项拟议的研究将为更好地治疗癌症从而改善公共健康的新疗法提供基础。

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.