Identification Of Key MicroRNAs Based On The Small RNAomics In Lipid Metabolism Of Sheep

Fat- and thin-tailed sheep are often considered as animal models to study the muscle development and adipose metabolic mechanism because of their differet phenotypes of fat tails, implying a difference in the regulation of adipose metabolism. MicroRNA (miRNA) is a kind of endogenous small non-coding RNA, regulates the expression at transcription level of genes by specifically combining with the target genes, participates in a series of physiological processes, including fat metabolism, as well as some pathological processes. Due to miRNA expression has the temporospatial specificity, studying the differential expression of miRNAs in sheep with different tail shape can identify more and better essential genes and metabolic pathways that related to the fat metabolism. Related results may lay a fundamental for further researching the fat metabolic mechanism and genetic improvement of ovine meat traits.In this research, high through-put sequencing technique was used to identify the expressed miRNAs in perirenal fat of Shanxi Dam Line (SHD), Small Tailed Han (STH) and Guangling Large tailed (GLT) sheep, respectively. Differentially expressioned miRANs in different breeds (strains) were screened out. In order to verifying the sequencing accuracy, abundance of 12 selected miRNAs in perirenal fat at 10-months of age was detected using qRT-PCR. The specific expression of five miRNAs in STH and GLT at 2,4,6,8,10, 12 months of age were analyzed, respectively, based on the qRT-PCR results. Influences of breed (strains), gender and month of age on the expression of miRNAs were also analyzed. Furthermore, target genes of miR-376e-3p were predicted using online software miRGator, miRDB and MicroCosm Targets. Biological functions of the target genes were analyzed through Gene Ontology (GO). The main results were as follows:1. Through the bioinformatics analysis of sequencing results,145 and 126 conserved ovine miRNAs were identified in perirenal fat libraries of SHD and STH, respectively. Using miReap,209 and 179 candidate novel miRNAs were also identified, respectively.2. By comparisons between differentially expressed miRNAs in three libraries i.e. constructed from GLT (GLTPEF), SHD (SHDPEF) and STH (STHPEF), the number of differentially expressed miRNAs was 105 between SHDPEF and STHPEF,103 between SHDPEF and GLTPEF, and 95 between GLTPEF and STHPEF, respectively.3. All 12 miRNAs that detected by qRT-PCR expressed in perirenal fat of three breeds (strain). The expression tendency of 11 miRNAs corresponded with the sequencing data, expect the opposite tendency of miR-383-3p. Among the 11 miRNAs, abundance of one novel and nine conserved miRNAs showed significant difference between breeds or strains (P<0.05). Above results can lay a foundation for further study the regulation of ovine miRNAs in adipose metabolism.4. The tendencies of five miRNAs in SDH, STH and GLT at 10 months of age were analyzed in this study. The results showed that, four miRNAs were detected as having the tendency of SHD*>STH*>GLT* (P<0.05). Inversely, miR-380-3p had the tendency of SHD*>GLT>STH. The expression of miR-376e-3p, miR-380-3p, miR-411a-3p and miR-487b-3p showed heterochrony during animal development. However, miR-494-3p expression was unrelated to development time. Besides, only miR-376e-3p showed significant difference between gender (P<0.05).5. MicroCosm Targets, miRDB and miRGator were used to predict the target genes of miR-376e-3p. Accordingly,744,465 and 319 target genes were obtained, respectively. In the three groups of targets genes, 25 common genes were screened out using the Venn method. The common genes were then analyzed using GO method, and the target genes of miR-376e-3p were enriched to four molecular functions and 14 biological processes.This research analyzed the expression of miRNAs in perirenal fat of sheep with different tail shapes, predicted the target genes and analyzed their biological functions. The related results can provide scientific basis for investigating the mechanism of miRNAs in regulating adipose metabolism, and the related methods provide references for identification and function study of miRNAs in regulating the genes that related to adipose metabolism.