Methods of systematic microRNA target validation and identification

描述（由申请人提供）：众所周知，microRNAs可以在多种细胞功能的调控中发挥基础作用。作为关键的基因调控因子，microrna通过转录后抑制机制发挥作用。越来越多的证据表明，microRNA表达的失调可能导致包括人类癌症在内的多种疾病。尽管在过去的几年中，在发现microRNA及其生物发生及其在许多细胞表型中的作用方面取得了重大进展，但由于单个microRNA可以有数百个靶点，因此人们尚未完全了解microRNA如何发挥其细胞功能。因此，鉴定microRNA靶点是理解microRNA在正常和疾病过程中介导的基因表达的分子机制的关键一步。目前，这在很大程度上依赖于计算机辅助算法，不幸的是，这些算法仍然无法提供microRNA调控网络的精确图像，因此预测的目标需要进一步的实验验证。很明显，靶标验证是我们努力解剖microRNA途径的瓶颈。在这个应用中，我们建议通过两种互补的方法来开发一种新的microRNA靶点验证和鉴定的选择方法。第一种方法是利用我们的前microRNA收集来确定microRNA/mRNA的相互作用，以对抗我们选择质粒中克隆的特定目标；第二种方法是使用针对特定microRNA的3'-UTR（非翻译区）文库来确定microRNA/mRNA的相互作用。我们相信我们的选择方法是创新的，简单的和强大的。这种方法的另一个好处是，除了种子序列的同源性外，我们还可以确定是否有任何新的特征，这可能有助于microRNA靶向的特异性。因此，本研究将极大地提高我们对microRNA靶向和基因调控的认识，提供一个有价值的研究工具。

DESCRIPTION (provided by applicant): It is well known that microRNAs could play a fundamental role in regulation of diverse cellular functions. As key gene regulators, microRNAs work through a posttranscriptional repression mechanism. Increasing evidence indicates that deregulation of microRNA expression could lead to a variety of disorders including human cancer. Although significant progress has been made in the past years in discovery of microRNAs and their biogenesis, and their role in many cellular phenotypes, it is not fully understood how microRNAs exert their cellular functions because a single microRNA can have hundreds of targets. Hence, identification of microRNA targets is a critical step toward understanding of molecular mechanisms of microRNA-mediated gene expression in normal and disease processes. Currently, this largely relies on computer-aided algorithms, which unfortunately are still unable to provide a precise picture of microRNA regulatory networks, and thus the predicted targets need further experimental validations. It is evident that target validation is a bottle neck in our effort to dissect microRNA pathways. In this application, we propose to develop a novel selection method for microRNA target validation and identification through two complementary approaches. The first approach is to determine microRNA/mRNA interactions using our pre-microRNA collection against a specific target cloned in our selection plasmid; the second approach is to determine microRNA/mRNA interactions using a 3'-UTR (untranslated region) library against a specific microRNA. We believe that our selection method is innovative, simple and powerful. An additional benefit of this method will allow us to determine whether there are any new features, besides the seed sequence homology, which could contribute to the specificity of microRNA targeting. Accordingly, this study will greatly enhance our understanding of microRNA targeting and gene regulation by providing a valuable research tool. PUBLIC HEALTH RELEVANCE: MicroRNAs are master gene regulators that work through a posttranscriptional repression mechanism. A single microRNA could target hundreds of targets. However, we do not have a full picture of microRNA/mRNA interactions yet. This study will develop a selection system to systematically determine microRNA/mRNA interactions. The success of this study will benefit microRNA research fields by providing a simple but powerful tool for microRNA target validation and identification.