Experimental Study Of Mir-260 Target Gene Identification And Dynamic Model

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.