Identification And Analysis Of MiRNAs In Skeletal Muscle Of Wuranke Sheep At Different Developmental Stages

Micro RNAs(miRNAs)are a class of endogenous noncoding RNAs of 18-25 nucleotides in length.Many miRNAs have been implicated in the regulation of skeletal muscle development.Wuranke sheep is an excellent mutton sheep breed,which has been bred for a long time in Inner Mongolia.To explore the roles of miRNAs underlying skeletal muscle development in Wuranke sheep,high-throughput sequencing was used to obtain miRNA and mRNA expression profiles in fetal,3-month-old,and 15-month-old Wuranke sheep biceps femoris.Based on an integrated analysis of miRNA and mRNA expression profiles,the differentially expressed miRNAs(DEMs)and their target genes involved in muscle development were screened out,and the regulatory networks were constructed.In addition,the tissues expression profiles of selected miRNAs were detected,and the relationship of their genetic variations and sheep growth traits were analyzed.The main results are as follows:1.In total,1,195 miRNAs were detected in nine small RNA libraries,including 988 known miRNAs and 207 novel miRNAs.The known miRNAs could be compared to 735 precursors for mammals in mi RBase v21.0.In the 3-month-old vs fetal comparison,474DEMs(213 upregulated and 261 downregulated)were identified,461 DEMs(186upregulated and 275 downregulated)were detected in the 15-month-old vs fetal comparison,and only 54 DEMs(8 upregulated and 46 downregulated)were in the15-month-old vs 3-month-old comparison.The expression trends of nine DEMs observed by RT-qPCR were consistent with those based on sequencing data,indicating that the sequencing results were reliable.2.A total of 24,959 genes were detected in nine mRNA libraries.Overall,the expression levels of these genes varied widely,and 91.3% of values were lower than 15 FPKM.In detail,we detected 6,783 differentially expressed genes(DEGs)(749upregulated and 6,034 downregulated)in the 3-month-old vs fetal comparison,7,407DEGs(795 upregulated and 6,612 downregulated)in the 15-month-old vs fetal comparison,and 78 DEGs(36 upregulated and 42 downregulated)in the 15-month-old vs3-month-old comparison.The expression trends of seven DEGs observed by RT-qPCR were consistent with those based on sequencing data,indicating that the sequencing results were reliable.3.Target Scan and mi Randa algorithms were used to identify miRNA binding sites to predict target genes of DEMs.Then,the results were combined with the mRNA sequencing data,and the miRNA–mRNA pairs with negative relationships were screened for further analysis.As a result,7,075 miRNA–mRNA pairs were obtained in the3-month-old vs fetal comparison(involving 382 miRNAs and 479 target genes),7,003 miRNA–mRNA pairs were obtained in 15-month-old vs fetal comparison(including 374 miRNAs and 519 target genes),and 19 miRNAs and 8 target genes formed 22 miRNA–mRNA pairs in the 15-month-old vs 3-month-old comparison.GO and KEGG enrichment analysis of target genes were performed.The results showed that some target genes were associated with skeletal muscle development,such as cell proliferation,cell differentiation,cell division,and myotube differentiation.The most significantly enriched pathway was the Notch signaling pathway(P<0.05),and the pathway with the largest number of target genes was the PI3K-Akt signaling pathway(P>0.05)in both3-month-old vs fetal and 15-month-old vs 3-month-old comparisons.In addition,“ribosome” was the only significantly enriched pathway(P<0.05)in the 15-month-old vs3-month-old comparison.4.Stem-loop RT-qPCR was used to analyze the tissues expression profiles of eight selected miRNAs.The results showed that oar-mi R-133,oar-mi R-299-5p and oar-mi R-1185-5p were higher expressed in skeletal muscle compared to other tissues or organs,of which oar-mi R-133 and oar-mi R-299-5p were the highest expressing in skeletal muscle at all the three developmental stages.5.Genetic variations of eight selected miRNAs were detected by direct sequencing in 90 blood samples from three sheep breeds with different meat production performances(Dorper sheep,Suffolk sheep and Wuranke sheep).A total of eight genetic variations were identified in five miRNAs,and all of them located in the upper or lower flanks of the precursor sequences.Polymorphic information content distribution was different in the three species.The polymorphisms of oar-mi R-133,oar-mi R-27 a,oar-mi R-29a?R+1 and oar-mi R-370-3p?R-2 genes were significantly relevant with some sheep growth traits.In this study,the miRNAs related with skeletal muscle development were screened by an integrated analysis of miRNA and mRNA expression profiles in the skeletal muscle of Wuranke sheep at different developmental stages,and their tissues expression profiles and genetic variations were further revealed,which will provide a reference for revealing the underlying functions of miRNAs in Wuranke sheep skeletal muscle development.