microRNA靶基因识别的系统生物学

microRNAs (miRNAs) are a class of small RNAs that inhibit the expression of specific protein-encoding genes in eukaryotes. A miRNA is typically predicted by computational methods to have hundreds of such target genes, although it is unclear how many of these predicted targets are actually regulated or the degree by which their expression is controlled by the miRNA in cells. We propose to study these questions using miR-1, miR-122, and miR-124 as representative miRNAs in 293T and HeLa cells, as testing three miRNAs in two different human cell culture models will help to generalize our findings. For each miRNA, we will select ~ 200 suspected targets based on online predictions, and for each such target, construct a reporter gene in which its putative miRNA-targeting sequence is incorporated into the 3’ untranslated region of a firefly luciferase gene. When the luciferase construct is transfected into cell cultures together with the miRNA, how much luciferase expression is affected (as compared to the transfection of a negative control RNA) will indicate whether the gene is indeed directly targeted by the miRNA and how well this gene is repressed by the miRNA in cells. We will then using computational methods to identify features in these target genes that can predict how well any gene is regulated by a miRNA. To further show that endogenous miRNAs function via the same mechanism, we will correlate our cell culture data with the reported mRNA and protein expression data in human cells and issues. If the endogenous target gene expression is negatively correlated with the degree of repression by miRNA as elucidated by our cell culture studies, it will be the first and strong evidence that miRNAs play a prominent role in differential expression of their target genes in vivo. Our results will further corroborate the functional significance of differential target repression by miRNAs, a property that has been generalized as relative specificity.

microRNAs（miRNA）是一类抑制蛋白编码靶基因表达的小RNA。据预测, 每个miRNA有数百个靶基因，但目前我们并不知道预测基因多少是正确的及它们受miRNA调节的程度。为回答这些问题，我们将用人类细胞模型研究三个代表性miRNA的功能。为每个miRNA, 构建一个疑似靶基因的荧光素酶报告基因的文库。报告基因转染细胞时, miRNA对荧光素酶的影响将显示对应的基因是否确为靶基因和它受miRNA调控的程度。然后, 计算方法将研究miRNA调节基因的决定因素。最后，为表明内源基因使用相同的机制，我们将进行人细胞和组织基因组数据的荟萃分析。若内源靶基因的表达与受miRNA抑制的程度呈负相关，这将表明miRNA在体内基因的差异性表达中发挥了突出作用，从而揭示miRNA差异性抑制靶基因(此特性已被概括为相对特异性)的功能意义。

本项目是关于microRNA（miRNA）功能机制的基础性研究。miRNA是真核生物中一类大量存在、极其重要的非编码RNA，通过互补序列结合靶基因mRNA并抑制其表达，从而广泛调控各种生理和病理过程。据预测, 每个miRNA有成百上千个靶基因，但目前并不知道预测基因多少是正确的及它们受miRNA调节的程度。本课题研究三个代表性miRNAs (人的miR-1、miR-122、miR-124)的功能。为每个miRNA，构建一个含200个软件预计（miRanda, Targetscan, PicTar）、疑似靶基因的荧光素酶报告基因的文库，然后，用报告基因和表达miRNA的质粒共转染293T细胞。通过miRNA对荧光素酶活性的影响检验对应的基因是否为该miRNA的靶基因和它受miRNA抑制的程度。对由此找到的一些靶基因，突变去掉miRNA结合位点，发现其不再受miRNA抑制，从而确认它们为靶基因。另外，部分结果也在HeLa细胞中进行验证。最后，用计算方法分析miRNA选择、调控靶基因的决定因素，并且，为表明细胞系的结果同样适用于内源基因，进行与人组织的转录组、蛋白组学数据的相关性分析，以检验miRNA是否在体内基因的差异性表达中发挥作用。主要结果和科学意义如下：（1）发现约60-80%的预测是可靠的，即其报告基因可被相应的miRNA所抑制。（2）绝大部分靶基因是第一次被直接确认，为今后研究miR-1、miR-122、miR-124和这些靶基因的功能提供了大量的全新数据、很好的实验基础和新的方向。（3）全面、系统地揭示了不同靶基因受miRNA抑制的程度各异。此特性已被概括为相对特异性，但在之前miRNA（和其它生化系统）的研究中普遍被忽视。（4）miRNA和靶基因mRNA的互补序列的结合能影响miRNA抑制的程度。（5）人内源基因的表达与受miRNA抑制的程度呈一定的负相关，从而首次揭示了miRNA调控着体内基因的差异性表达，即相对特异性的功能意义。