Expression Profiles Of MiRNA And MRNA In Chicken Hypothalamus And Its Bioinformatics Analysis

MicroRNAs （miRNAs） are small non-coding regulatory RNAs with 19–24 nucleotides （nt）, which play an important role in the post-transcriptional regulation of gene expression during development, and in other biological processes. Most of these miRNAs have been identified from whole embryos, somites and primary fibroblast cells. However, relatively little work has been done to study miRNA expression and miRNA function in the later stages of development in the chicken. miRNA operate highly complex regulatory networks. Current estimates indicate that each miRNA potentially could have hundreds of target genes, and each gene can combine with numerous miRNAs. Former studies mainly focus on the miRNA expression profiling,then predict the target genes, which may not completely illustrate the whole gene regulation network.Therefore, identification of new miRNAs in different organisms and phases is a critical step in the study of the biological functions of miRNAs.In this subject, expression profile of miRNA and mRNA in hypothalamus in different developmental stages chicken are to be studied by solexa sequencing technology. The expermental results are to be verified by real-time quantitative PCR, and the corresponding binding sites of miRNA target genes are to be analyzed through bioinformatics.we explore the gene and miRNAs expression profile in hypothalamus,to illustrate the mechanism of miRNAs and genes involved in hypothalamus.Simultaneously,we try to use the comparative genomics and systems biology to find out the mechanism of the possible interaction between miRNAs and genes, to clarify the regulation mechanism of miRNA in animal growth and development and meat conformation, to find out new target genes for gene engineering breeding in meat quality, to offer new idea and useful clue for molecular improvement in meat quality.1、Differentially expressed miRNAs in two phases of chicken by Deep sequencing In this study, we used Deep sequencing technologies to identify the chicken miRNAs, and we compared the characteristics and expression patterns of miRNA in the hypothalamus of 1-day-old and 36-weeks chickens, and KEGG （Kyoto Encyclopedia of Genes and Genomes） pathway analysis of predicted target genes to determine the global biological functions of different miRNA.The number of RNAs with high-quality reads was different in the two RNA libraries. For example, in the library from 1-day-old chickens, almost half of the high-quality reads （44.35%） were 22nt in length, followed by 23nt （26.37%）; however, in the library from 36-weeks chickens, the length distribution peaked at 23nt （23.06%）, followed by 22nt （20.24%）. The proportions of other small RNA types in the two libraries were significantly different. We found 179 miRNAs that had significant differences in expression between the two libraries （174 miRNA that had more than one-fold difference, p<0.01; 5 miRNA, 0.01<p<0.05）; 157 of them appeared to be down-regulated from the 1-day-old to the 36-weeks chicken, while 22 seemed to be up-regulated in the same order.In this study, we identified 363 potential novel miRNAs that corresponded collectively to 363 independent genomic loci. In total, 85 miRNAs were present in the two libraries, white 112 and 166 miRNAs were expressed specifically in the 1-day-old and 36-weeks hypothalamus, respectively.The 4362 target genes were predicted by RNAhyrid which are involved with KEGG pathway and GO overrepresentation. Target genes were mostly existed in the pathway of the cell and renal cell metabolic and regulation cell metabolic using KEGG functiongal annotations. The gene ontology category indicated that these target genes mainly participate in cellular process,biological regulation, regulation of cellular process, regulation of biological process and metabolic process.2、Expressions of miRNA gene in different developmental stages and tissues The expression profiles of miR-9, miR-181a and miR-92 in 11 normal tissues of two stage chicken were studied by real-time RT-PCR using stem-loop RT-primers. TargetScan 5.1 and PicTar were used to predict the target genes of miR-9, miR-181a and miR-92 and the intersection of the results as gene set was analyzed by KEGG analysis.To confirmation of differentially expressed miRNAs. The real-time RT-PCR were used to confirm the expression pattern of differentially expressed miRNAs in chicken hypothalamus. The change directions of differentially expressed miRNAs between SYBR Green qPCR and deep sequence analysis in four miRNAs was general consistency, although the expressed miRNAs level was different between SYBR Green qPCR and deep sequence analysis. The results of SYBR Green PCR showed that the expressions of miR-9、miR-181a and miR-92 was significantly different in different tissues and different stage.The 160,137,157 target genes were predicted by algorithms PicTar and TargetScan which are involved with KEGG pathway. Target genes of miR-9 were mostly existed in the pathway of the regulation of cytoskeleton and Renal cell carcinoma etc using KEGG functiongal annotations. miR-181a target genes were mainly involved in neurotrophin signaling pathway, long-term potentiation, Aldosterone-regulated sodium reabsorption, T cell receptor signaling pathway and Oocyte meiosis. miR-92 target genes were mainly involved in Long-term potentiation, Type II diabetes mellitus, MAPK signaling pathway, Amino sugar and nucleotide sugar metabolism These experimental results would provide a beneficial reference for further study of miRNA in brain on the regulation in animal metabolic and development.3、Transcriptome profiling of developing chicken by deep-sequencingThe result that the distribution of total and distinct tag counts over different tag abundance categories showed very similar tendencies for two libraries. Among the distinct tags, 4.81% and 5.18% copy number were expressed specifically in the 1-day-old and 36-weeks hypothalamus, respectively. White as 80% of the tags were present between 2 and 50 copies, and more than 40% of the transcripts were 2–5 copies.The differentially expressed mRNA in the hypothalamus of 1-day-old and 36-weeks chickens were screened by deep sequencing. The results showed 1143 mRNAs were down-regulated and 413 mRNAs were up-regulated in 36-weeks hypothalamus. 50 and 17 were expressed specifically in the 1-day-old and 36-weeks hypothalamus, respectively.Different genes were mostly existed in the pathway of metabolic and regulation cell metabolic using KEGG functiongal annotations and ontology category.4、Clone and bioinformatics analysis of chicken PMCH gene In this study, The full-length cDNA was amplified by RT-PCR, 3’-RACE and 5’-RACE from chicken hypothalamus. A cDNA of 803 bp sequence was obtained, and was submitted on GenBank （accession number: HM853641）. The exon 1 of chicken PMCH gene contained the translation start site. The full-length cDNA contained an ORF of 492 bp, which coded for a protein of 163 amino acids. The 5’-untranslated region （-UTR） was 68 bp and was followed by an ATG initiation codon. The stop codon TGA was at the position 558-560 bp. The putative polyadenylation signal AATAAA was found 20 bp upstream from the 14-nucleotide poly （A） tail, which suggested a 243 bp long of 3’-UTR in the chicken PMCH full-length mRNA. The introns and exons were obtained by contig analysis and GT-AG rule. Chicken PMCH gene is composed of 3 exons, which is consistent with those of human and bovine. Its sequence was shorter than the predicted PMCH （XM₄16324.1） at the 5’end. This was due to the mistaken prediction of one of exon1. A nucleotide BLAST search showed that full-length cDNA was homologous to mammalian PMCH genes. It was 65.95%, 62.69%, 65.19%, 66.54% and 59.62% identical to the cDNA sequences of human,mouse,cattle,Pan troglodytes and rat PMCH, respectively. Otherwise, the chicken shared 52.07%, 50.30%, 52.07%, 50.89% and 50.30% homology with human,mouse,cattle,Pan troglodytes and rat in the amino acid sequence of PMHC, respectively.The PMCH protein sequence is predicted to have several functional domains, including pro-MCH,CSP,IL7,XPGI and some low complexity sequence. It has 8 phosphorylation sites and no signal peptide sequence. There is a hydrophobic region in 140-160aa.Ten function motifs has be found in the amino acid sequence. Three predicted microRNA targeting site was found in the 3′untranslated region of chicken PMCH mRNA.In a word, our work study the expression profile of miRNA and mRNA in chicken hypothalamus of different developmental stages by deep sequencing technology, firstly. And we provide a thorough account of the miRNA transcriptome in chicken hypothalamus tissue. Imultaneously, we use the systems biology to study interaction between miRNAs and genes. Meanwhile, our data also provide new insights into the biological role of miR-9, miR-181a and miR-92.We first study to clone and analysized of chicken PMCH gene.