【作者】 刘婷；

【导师】 贾亚；

【作者基本信息】 华中师范大学 ， 理论物理， 2017， 硕士

【摘要】 microRNA(miRNA)是广泛存在于细胞中的一类最小的功能非编码RNA(non-codingRNA,ncRNA)。其生物学功能,主要表现为对靶基因(主要是信使RNA,mRNA)的调控。确定miRNA靶基因的方法有分子生物实验和信息学分析两种。实验方法是研究miRNA调控后mRNA的水平及其蛋白变化;生物信息学方法则是依据已被实验证实的miRNA与其靶基因序列之间的相互作用规律设计成的软件进行大数据分析,在基因库中寻找miRNA的可能调控的目的基因。实验研究表明:mir-260突变体能够加快秀丽隐杆线虫的衰老进程。可能的分子机制是mir-260通过间接影响基因jnk-1、eat-2的表达量来调控线虫的衰老进程。本文以mir-260为研究对象,以生物信息学预测和分子实验检测相结合的方法,研究mir-260对与衰老相关基因的直接调控。并结合实验数据,改进了基因调控的动力学模型。主要工作如下:在实验上,利用生物信息学方法(mircoRNA、mirSOM)预测mir-260的靶基因,并筛选出其中5个已报道与寿命有关的基因:coq-2、nuo-4、rps-10、tps-1、atp-3。进行分子实验,构建含有mir-260和上述靶基因的质粒,并将重组质粒转染到293T细胞,再利用双荧光素酶报告基因方法检测5个靶基因的荧光蛋白在不同时间的表达量。实验结果表明:1,coq-2、nuo-4、rps-10、tps-1、atp-3 是 mir-260 靶基因;2,mir-260对靶基因的调控在转染后的36到60小时最强。在理论上,本文考虑了细胞内生物反应的累积性,改进了 miRNA与mRNA相互作用的动力学模型,并利用改进后的模型,拟合实验数据。更多还原

【Abstract】 microRNAs(miRNAs)are a class of minimal functional non-coding RNAs in cells.Its biological function,mainly for the target gene(mainly messenger RNA,mRNA)regulation.The methods used to determine the target genes of miRNA are molecular biology experments and bioinformatics.The biological experimental approach is to study the level of mRNA and its protein changes after miRNA regulation.The bioinformatics approach is based on the regularity of the interaction between the proven miRNA and the target gene sequence,followed by several commonly used principles to design some kind of software for big data analysis and searching for the possible target genes of miRNA in gene pool.Experimental studies have shown that:mir-260 mutants can accelerate the aging process of C.elegans.The possible molecular mechanism is that mir-260 regulates the senescence of nematodes by indirect influencing the expression of gene jnk-1 and eat-2.In this paper,we used mir-260 as the research object and applied the method of combining bioinformatics prediction and molecular experiment to study the direct regulation of mir-260 on senescence related genes.Based on the experimental data,improved the dynamic model of gene regulation.The main work is briefly iIlustrated as follows:In our experiment,we first used the bioinformatics method(mircoRNA、mirSOM)to find mir-260 regulatory genes and screened out five key genes coq-2、nuo-4、rps-10、tps-1、atp-3 which are have been reported related to the senescence of the C.elegans.We carryed out the molecular experiments to construct plasmids containing mir-260 and the target gene and the recombinant plasmid was transfected into 293T cells,then using the dual luciferase reporter assay for detecting protein expression of 5 target genes in different time.Experimental results show:1,coq-2,nuo-4,rps-10,tps-1,atp-3 are mir-260 target genes.The regulation of target gene by mir-260 was strongest in 36 to 60 hours after transfection.Theoretically,we consider the accumulation of biological response.We improved the existing model for the interaction between miRNA and the mRNA,then use the improved model to fit the experimental data.更多还原