Screening And Functional Study Of Immune Related MicroRNA In Megalobrama Amblycephala After Aeromonas Hydrophila Infection

Blunt snout bream(Megalobrama amblycephala),commonly known as Wuchang fish,is an important economic fish species of freshwater aquaculture in China.In recent years,due to the expansion of aquaculture density and deterioration of the ecological environment,various kinds of diseases occur.Among these diseases,the bacterial septicemia caused by Aeromonas hydrophila emerged as one of the most harmful to the development of M.amblycephala aquaculture in China.So far,there is no effective prevention or treatment measure for the bacterial septicemia.MicroRNAs(miRNAs)are a class of small non-coding single-stranded RNAs that play important roles in the immune response.Unfortunately,the characterization of M.amblycephala miRNAs in response to A.hydrophila infection has not been performed.To better understand the biological significance of miRNA in M.amblycephala after A.hydrophila infection,three small RNA libraries were constructed and analyzed from the liver at 0 h(non-infection),4 h and 24 h post infection(poi)using Illumina deep sequencing technology.MiRNAs related to the immune response of M.amblycephala after A.hydrophila infection were identified.Subsequently,the functional analysis of the immune-related miRNAs and the regulation between these miRNAs and their target genes were analyzed.The main results are as follows:1.Three small RNA libraries were constructed from the liver of M.amblycephala at 0 h(non-infection),4 h and 24 h post infection(poi)using high throughput sequencing technology and a total of 11,244,207,9,212,958 and 7,939,157 clean reads were obtained from them,respectively.Bioinformatics analysis identified 171 conserved miRNAs and 62 putative novel miRNAs.The existence of ten randomly selected novel miRNAs was validated by RT-PCR.Pairwise comparison of the three libraries revealed that 61 and 44 miRNAs were significantly differentially expressed at 4 h and 24 h poi,respectively.Furthermore,the expression profiles of nine randomly selected miRNAs were validated by qRT-PCR,and the analyses of target prediction,Gene Ontology(GO)enrichment and Kyoto Encylopedia of Genes and Genomes(KEGG)indicated that a variety of biological pathways could be affected by A.hydrophila infection.Of the predicted target genes,94 immune-related genes were identified.The expression patterns of randomly selected immune-related miRNAs in the liver of M.amblycephala after A.hydrophila infection were detected by qRT-PCR.The relationship between these mi RNAs and their corresponding target genes was validated by dual luciferase reporter assay.These results suggested that the differentially expressed miRNAs may be involved in the immune response to bacterial invasion by regulating the corresponding target genes.These results would expand our knowledge of the role of miRNAs in the immune response of M.amblycephala to A.hydrophila infection,and facilitate the development of effective strategies against A.hydrophila infection in M.amblycephala and selection of molecular markers for resistance breeding.2.The targets prediction results of differentially expressed miRNAs showed that miR-375 was collectively predicted to bind to transferrin(TF)and transferrin receptor(TFR)genes.The qRT-PCR results showed that the relative expressions of TF and TFR were significantly up-regulated,while miR-375 was down-regulated in the liver of M.amblycephala after A.hydrophila infection.The expressions of miR-375 and its target genes showed an inverse relationship.Finally,our results demonstrated that the 3' UTR of TF and TFR is targeted by miR-375,and miR-375 can repress the endogenous mRNA expression of TF and TFR by over expression experiment and dual luciferase reporter assay.Given the important roles of TF and TFR in iron homeostasis,we speculated that miR-375 may play an important role in iron homeostasis and resistance to bacterial invasion in M.amblycephala.3.The targets prediction results of differentially expressed miRNAs showed that miR-146 a,miR-29 a and miR-457b-5p were collectively predicted to bind to TRAF6 gene.The results of dual luciferase reporter assay demonstrated that miR-146 a and miR-29 a can target the 3'UTR of TRAF6 gene.Subsequently,the expression level of TRAF6 was inhibited or up-regulated by over expression or inhibitor miR-146 a and miR-29 a,respectively.These results confirmed that TRAF6 was a direct target of miR-146 a and miR-29 a.After infected by A.hydrophila,the expressions of miR-146 a,miR-29 a and TRAF6 were significantly increased in the liver,spleen and kidney,and all of them increased firstly and then decreased.Similarly,in CIK cells stimulated with lipopolysaccharides(LPS),the expressions of miR-146 a and miR-29 a were significantly up-regulated and reached the highest at 12 h,and finally returned to normal level at 48 h.These results suggested that miR-146 a and miR-29 a play important roles in the immune response to bacterial infection.Over expression of miR-146 a and miR-29 a in CIK cells could inhibit the downstream immune-related genes of Toll like receptor(TLR)/ NF-?B pathway,such as NF-?B p65,NF-?B p52,interleukin-6(IL-6)and tumor necrosis factor ?(TNF-?).Oppositely,in CIK cells stimulated with LPS,the downstream immune-related genes were activated when inhibiting the expression of mi R-146 a and miR-29 a.We speculated that miR-146 a and mi R-29 may be involved in the regulation of TLR/ NF-?B pathway and play important role in innate immune response in fish.