Identification And Function Prediction Of MicroRNA Involved In Anti-disease And Immune Responsesin Channel Catfish(Ietalurus Punetaus)

MicroRNAs(miRNAs) are a class of endogenous non-coding small RNA molecules widely exists in animals and plants. Mi RNAs mainly involve in post-transcriptional gene regulation and plays an important regulatory role in many life activities include organism development, cell activity, regulation of immune function and disease development. In studies of miRNAs in human and other animals, there are a lot of reports and increasing depth day by day. Fish as important vertebrates, are relatively lack of reports of miRNAs, especially in the respect of regulation on anti-disease and immune research. In order to study the effect in the immune regulation of fish miRNAs, this paper studied miRNAs involved in immune system of channel catfish which is the representative in teleosts. Firstly, the expressions of nine immune-related miRNAs and six cytokines in immune tissues of channel catfish under immune stimulation of lipopolysaccharide(LPS) and β-glucan were studied. On this basis, the second generation of high-throughput sequencing and bioinformatics analysis were applied to constructed miRNAs expression profile of channel catfish in immune tissues before and after Vibrio mimicus infection, and predict the functions of differentially expressed miRNAs. The results not only provided more information on anti-disease and immune regulatory functions of miRNAs, but also built a good foundation for future stuies on miRNAs function. In this paper, the study was constituted by two parts.Part one: the expressions of immune-related miRNAs and cytokines in Channel Catfish under immuno-stimulatory of LPS and β-glucan were detected.40 channel catfish with an average body weight of 18.48 ± 0.20 g were randomly divided into two groups. Fish in control group were injected with PBS and those in test group were injected with LPS(5 mg/ml) 0.1 ml / tail. After 24 hours, samples of major immune organs including liver, head kidney and spleen were collected. 240 channel catfish with an average body weight of 2.20 ± 0.02 g were randomly divided into two groups for feeding with basal diet or β-glucan(1 g/kg) diet for 30 days. After feeding, samples of liver, head kidney and spleen were collected to analyze immune-related miRNAs and six immune-related cytokines by reverse transcription quantitative PCR. The results showed that the expressions of nine miRNAs immune-related in the liver, head kidney, spleen were mainly significantly upregulated, and the greater expression of miRNAs were induced by LPS. Six cytokines were differentially expressed in the liver, head kidney, spleen one or a few tissues, the expressions induced by β-glucan were mainly downregulated, while the expressions induced by LPS were mainly upregulated.Part two: miRNAs expression profiling construct and the target gene analysis for Channel Catfish before and after Vibrio mimicus infected80 channel catfish with an average body weight of 170.08±23.75 g were randomly divided into two groups for injection with PBS or Vibrio mimicus(6.78×107 /ml) 0.1 ml tail respectively. After 48 hours, samples of head kidney and spleen were collected and frozen in liquid nitrogen for later extraction of total RNA. The RNA were reverse transcribed into c DNA after testing the quality of total RNA uptostandard, then through Hiseq high-throughput sequencing technology and bioinformatics analysis, four c DNA libraries were constructed for head kidney, spleen tissue in Channel Catfish before and after Vibrio mimicus infected. The sequenced reads were annotated according to the reference genome of related species of Astyanax mexicanus, and compared to the known miRNAs of all the animals in the mi RBase database. 1504 and 1658 conserved miRNAs and25 and 27 new miRNAs were identified in head kidney before and after infection, respectively. And 1407 and 1258 conserved miRNAs and 23 and 15 new miRNAs were identified in spleen before and after infection, respectively. Comparing miRNAs expression level before and after injection with Vibrio mimicus by Expdiff, 502 conserved miRNAs and 10 new miRNAs in head kidney as well as 281 conserved miRNAs and 6 new miRNAs in spleen were differentially expressed. After analyzing these differentially expressed conserved and new miRNAs and predicting the target genes, these target genes was performed functional classification enrichment and metabolic pathway analysis, and it was found that a variety of target genes enriched in immune-related pathways.In conclusion, the results showed that nine of immune-related miRNAs may play an important role in the regulation of immune responses to the stimulations of LPS and β-glucan. IL-1, TNF-α, IFN-γ, TLR2 and other cytokines may be involved in immune regulation process of miRNAs. Mi RNAs in head kidney and spleen were significantly differentially expressed after the infection of Vibrio mimicus in channel catfish. The target genes of miRNAs which were differentially expressed mainly enriched in immune-related pathways. This dissertation provided basic information for the studies on immune-related miRNAs as well as the immune regulation mechanism of miRNAs in fish.