Long non-coding RNAs (lncRNA) and microRNAs (miRNA) are new found classes of non-coding RNAs (ncRNAs) that are not translated into proteins but regulate various cellular and biological processes. In this study, we conducted a transcriptomic analysis of ncRNA and mRNA expression in Aohan fine wool sheep (AFWS) at different growth stages (embryonic day 90, embryonic day 120, and the day of birth), and explored their relationship with wool follicle growth. In total, 461 lncRNAs, 106 miRNAs, and 1,009 mRNAs were found to be differentially expressed during the three stages of wool follicle development. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to clarify the roles of the differentially expressed lncRNA, miRNA, and mRNA in the different stages of wool follicle development. Quantitative real-time PCR (qRT-PCR) was used to validate the results of RNA-seq analysis. lncRNA (MSTRG.223165) was found to act as a competing endogenous RNA (ceRNA) and may participate in wool follicle development by acting as an miR-21 sponge. Network prediction implicated the MSTRG.223165-miR-21-SOX6 axis in the wool follicle development. The targeting relationships of miR-21 with SOX6 and MSTRG.223165 were validated in dual-luciferase assays. This is the first report indicating the association of the lncRNA–miRNA–mRNA network with wool follicle development in AFWS. This study provides new insights into the regulation of the wool follicle growth and represents a solid foundation for wool sheep breeding programs.

Seasonal estrus is a critical limiting factor of animal fecundity, and it involves changes in both ovarian biology and hormone secretion in different seasons. Previous studies indicate that two classes of small RNAs (miRNAs and piRNAs) play important regulatory roles in ovarian biology. To understand the roles of small RNA-mediated post-transcriptional regulation in ovine seasonal estrus, the variation in expression patterns of ovarian small RNAs during anestrus and the breeding season were analyzed using Solexa sequencing technology. In addition, reproductive hormone levels were determined during ovine anestrus and the breeding season. A total of 483 miRNAs (including 97 known, 369 conserved and 17 predicated novel miRNAs), which belong to 183 different miRNA families, were identified in ovaries of Tan sheep and Small Tail Han (STH) sheep. Compared with the three stages of the breeding season, 25 shared significantly differentially expressed (including 19 up- and six down-regulated) miRNAs were identified in ovine anestrus. KEGG Pathway analysis revealed that the target genes for some of the differentially expressed miRNAs were involved in reproductive hormone related pathways (e.g. steroid biosynthesis, androgen and estrogen metabolism and GnRH signaling pathway) as well as follicular/luteal development related pathways. Moreover, the expression of the differentially expressed miRNAs and most of their target genes were negatively correlated in the above pathways. Furthermore, the levels of estrogen, progesterone and LH in ovine anestrus were significantly lower than those in the breeding season. Combining the results of pathway enrichment analysis, expression of target genes and hormone measurement, we suggest that these differentially expressed miRNAs in anestrus might participate in attenuation of ovarian activity by regulating the above pathways. Besides miRNAs, a large and unexpectedly diverse set of piRNAs were also identified. The miRNA profiles of ovine ovaries in anestrus were presented for the first time. The identification and characterization of miRNAs that are differentially expressed between ovine anestrus and the breeding season will help understanding of the role of miRNAs in the regulation of seasonal estrus, and provides candidates for determining miRNAs which could be potentially used to regulate ovine seasonal estrus.

Intramuscular fat (IMF) is one of the most critical parameters affecting meat quality and mainly affected by genetic factors. MicroRNA as an important regulatory factor, which is still a lack of research in the development of sheep IMF deposition. We used RNA sequencing (RNA-seq) and cell-level validation to explore the role of miRNA in IMF deposition. As for this purpose, longissimus thoracis et lumborum (LTL) samples of 2 month-old (Mth-2) and 12 months-old (Mth-12) Aohan fine-wool sheep (AFWS) were used to identified miRNAs expression. We found 59 differentially expressed miRNAs (DE-miRNA) between these age groups and predicted their 1,796 target genes. KEGG functional enrichment analysis revealed eight pathways involved in lipid metabolism-related processes, including fatty acid elongation and the AMPK signaling pathway. A highly expressed DE-miRNA, miR-193a-5p, was found to serve a function in 3T3-L1 preadipocyte differentiation. Luciferase assay demonstrated that miR-193a-5p directly binds to the 3′-UTR region of ACAA2. By constructing mimics and inhibitor vector transfecting into 3T3-L1 cells to explore the effect of miR-193a-5p on cell proliferation and differentiation, we demonstrated that overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte proliferation, as evidenced by decreased mRNA and protein expression of CDK4 and CyclinB. CCK-8 assay showed that miR-193a-5p significantly inhibited cell proliferation. Similarly, the overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte differentiation and adipocyte-specific molecular markers’ expression, leading to a decrease in PPARγ and C/EBPα and ACAA2. Inhibition of miR-193a-5p had the opposite effects. Our study lists the miRNAs associated with intramuscular lipid deposition in sheep and their potential targets, striving to improve sheep meat quality.

Background: Aohan fine wool sheep (AFWS) is a historically bred fine wool sheep, cultivated in China. The wool has excellent quality and good textile performance. Investigating the molecular mechanisms that regulate wool growth is important to improve wool quality and yield. Circular RNAs (circRNAs) are widely expressed non-coding RNAs that can act as competitive endogenous RNAs (ceRNAs) to bind to miRNAs. Although circRNAs have been studied in many fields, research on their activity in sheep wool follicles is limited. To understand the regulation of circRNAs in the growth of fine wool in sheep, we used RNA-Seq to identify circRNAs in sheep shoulder skin samples at three developmental stages: embryonic day 90 (E90d), embryonic day 120 (E120d), and at birth (Birth). Results: We identified 8753 circRNAs and found that 918 were differentially-expressed. We then analyzed the classification and characteristic of the circRNAs in sheep shoulder skin. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we identified the source genes of circRNAs, which were mainly enriched in cellular component organization, regulation of primary metabolic processes, tight junctions, and the cGMP-PKG and AMPK signaling pathways. In addition, we predicted interactions between 17 circRNAs and eight miRNAs, using miRanda software. Based on the significant pathways, we speculate that circ_0005720, circ_0001754, circ_0008036, circ_0004032, circ_0005174, circ_0005519, and circ_0007826 might play an important role in regulating wool follicle growth in AFWS. Seven circRNAs were randomly selected to validate the RNA-Seq results, using qRT-PCR. Conclusion: Our results provide more information about circRNAs regulation of wool follicle development in AFWS, and establish a solid foundation for future research.

Background: Intramuscular fat (IMF) content has become one of the most important indicators for measuring meat quality, and levels of IMF are affected by various genes. Long non-coding RNAs (lncRNAs) are widely expressed non-coding RNAs that play an important regulatory role in a variety of biological processes; however, research on the lncRNAs involved in sheep IMF deposition is still in its infancy. Aohan fine-wool sheep (AFWS), one of China’s most important meat-hair, dual-purpose sheep breed, provides a great model for studying the role of lncRNAs in the regulation of IMF deposition. We identified lncRNAs by RNA sequencing in Longissimus thoracis et lumborum (LTL) samples of sheep at two ages: 2 months (Mth-2) and 12 months (Mth-12).Results: We identified a total of 26,247 genes and 6935 novel lncRNAs in LTL samples of sheep. Among these, 199 mRNAs and 61 lncRNAs were differentially expressed. We then compared the structural characteristics of lncRNAs and mRNAs. We obtained target genes of differentially expressed lncRNAs (DELs) and performed enrichment analyses using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that target mRNAs were enriched in metabolic processes and developmental pathways. One pathway was significantly enriched, namely tight junction. Based on the analysis of critical target genes, we obtained seven candidate lncRNAs that potentially regulated lipid deposition and constructed a lncRNA-mRNA co-expression network that included MSTRG.4051.3-FZD4, MSTRG.16157.3-ULK1,MSTRG.21053.3-PAQR3, MSTRG.19941.2-TPI1,MSTRG.12864.1-FHL1, MSTRG.2469.2-EXOC6and MSTRG.21381.1-NCOA1. We speculated that these candidate lncRNAs might play a role by regulating the expression of target genes. We randomly selected five mRNAs and five lncRNAs to verify the accuracy of the sequencing data by qRT-PCR.Conclusions: Our study identified the differentially expressed mRNAs and lncRNAs during intramuscular lipid deposition in Aohan fine-wool sheep. The work may widen the knowledge about the annotation of the sheep genome and provide a working basis for investigating intramuscular fat deposition in sheep.

Aohan fine-wool sheep (AFWS) is a high-quality fine-wool sheep breed that supplies wool and meat. Research is needed on the molecular mechanism behind intramuscular fat (IMF) deposition that greatly improves mutton quality. The widely expressed non-coding RNA is physiologically used in roles such as competitive endogenous RNA (ceRNA) that includes circular RNAs (circRNAs). Although circRNAs were studied in many fields, little research was devoted to IMF in sheep. We used the longissimus dorsi muscle of 2 and 12-month-old AWFS as research material to identify circRNAs related to IMF deposition in these sheep by RNA-seq screening for differentially expressed circRNAs in the two age groups. A total of 11,565 candidate circRNAs were identified, of which the 104 differentially expressed circRNAs in the two age groups were analyzed. Enrichment analysis was performed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. The enriched pathways included lipid transport (GO:0006869), negative regulation of canonical Wnt signaling pathway (GO:0090090), fat digestion and absorption (ko04975), and sphingolipid metabolism (ko00600). The differentially expressed circRNAs included ciRNA455, circRNA9086, circRNA7445, circRNA4557, and others. The source genes involved in these pathways might regulate IMF deposition. We used the TargetScan and miRanda software for interaction analysis, and a network diagram of circRNA-miRNA interactions was created. CircRNA455-miR-127, circRNA455-miR-29a, circRNA455-miR-103, circRNA4557-mir149-5p, and circRNA2440-mir-23a might be involved in the IMF deposition process. The targeting relationship of circRNA4557-miR-149-5p was verified by a dual-luciferase reporter assay. The RT-qPCR results of seven randomly selected circRNAs were consistent with the sequencing results. This study provides additional information on circRNA regulation of IMF deposition in AFWS and is a useful resource for future research on this sheep breed.