Identification And Expression Of MiRNA In BmN Cells And Screeing For Target Genes Of The Overexpressed MiRNAs

MicroRNAs(miRNAs) are the family of non-conding, small RNA molecules of21-25nucleotides in length and universally exist in Caenorhabditis elegans, Drosophila, Bombyx mori,human and Arabidopsis thaliana and other organisms. miRNAs participate in a variety ofphysiological and pathological process including cell proliferation, differentiation, apoptosis,signal transduction and tumorigenesis by repressing mRNA translation or cleaving the targetmRNA. In present, a lot of miRNAs were identified by experimental and computationalapproaches, including489pre-miRNAs and567mature miRNAs in bmobyx mori, but theirfunctions remain unknown. Research evidence indicates that the way of gain-of-function andloss-of-function can be obtained by ectopic miRNA expression and miRNA antagonism,respectively, were used to identify putative miRNA-targeted genes and its functions. Until now,there was little progress about miRNA function in Bmobyx mori.To carry out the research of miRNA targeted genes and its functions, we overexpressed thelevel of miRNA in BmN cells by transfected with miRNA eukaryotic expression vector andmiRNA mimics. Firstly, we identified the endogenous expression of miRNAs in BmN cells bymicroarray and found that only73miRNAs could be detected. Then four low abundance orundected miRNA that play an important role in gene regulation of other species, were selected toexpress in BmN cells. EGFPse quences was inserted into pIEx-1vector harboring baculovirusie1promoter and hr5enhancer to construct recombinant miRNA expression vector pIEx-1-EGFP. Then, pri-mir-1a/8/14/133sequences were inserted into the downstream of EGFP geneof pIEx-1-EGFP vector to construct eularyotic miRNA expression vector pIEx-1-EGFP-pri-mir-1a/8/14/133, respectively. Four miRNA expression vector pIEx-1-EGFP-pri-mir-1a/8/14/133were transfected into BmN cells and used for expressing bmo-miR-1a/8/14/133. Stem-loop RT-PCR analysis that the relative abundance of bmo-miR-1a,bmo-miR-8,bmo-miR-14,bmo-miR-133in BmN cells transfected with pIEx-1-EGFP-pri-mir-1a/8/14/133is as32,4.4,6.6and3910times as that BmN cells transfected with the control vector pIEx-1-EGFP, respectively. At thesame time, the chemical synthesis miRNA mimics were successfully transfected into BmN cellsand Stem-loop RT-PCR analysis showed that the relative abundance of bmo-miR-1a/8/14inBmN cells transfected with bmo-miR-1a/8/14mimics is as56,39and2933times as that theBmN cells transfected with negative control mimics. Northern blot results comfirmed that therecombinant plasmid pIEx-1-EGFP-pri-mir-133was effectively processed into mature bmo-miR-133. From the above, we successfully overexpressed bmo-miR-14/133in BmN cells. In order tocarry out the screening of bmo-miR-14/133target genes and its function, we analysized the effecton mRNA and protein in BmN cells overexpressed bmo-miR-14/133by expression profiling and two-dimensional electrophoresis. The results showed that bmo-miR-14changed the expressionlevel of some mRNA and protein in BmN cells, there are13mRNA level differential genesincluding six of them are dowenregulated; there are15gene of downregulated protein expressionlevel including riblsomal protein, translation initiation faction and so on. Similarity, bmo-miR-133changed the expression level of mRNA and protein in BmN cells, there are9differentialexpressin genes including eight of them are downregulated;14-3-3protein were downregulatedby2-D and mass spectrometry. miRNA target sites analysis showed that the3’UTR ofdownregulated gene of mRNA level and protein expression exist in potential mRNA bindingsites, it is the potential target genes of miRNA.Then the further these binding sites validationexperiments in vitro by the dual luciferase are being arranged. The present work lays afoundation for the gene regulation function of bmo-miR-14/133in Bmobyx mori.