The Identification and Characteristics of miRNAs Related to Cashmere Fiber Traits in Skin Tissue of Cashmere Goats.

microRNAs (miRNAs) are involved in the regulation of biological phenomena by down-regulating the expression of mRNAs. In this study, Liaoning cashmere (LC) goats (n = 6) and Ziwuling black (ZB) goats (n = 6) with different cashmere fiber production performances were selected. We supposed that miRNAs are responsible for the cashmere fiber trait differences. To test the hypothesis, the expression profiles of miRNAs from the skin tissue of the two caprine breeds were compared using small RNA sequencing (RNA-seq). A total of 1293 miRNAs were expressed in the caprine skin samples, including 399 known caprine miRNAs, 691 known species-conserved miRNAs, and 203 novel miRNAs. Compared with ZB goats, 112 up-regulated miRNAs, and 32 down-regulated miRNAs were found in LC goats. The target genes of the differentially expressed miRNAs were remarkably concentrated on some terms and pathways associated with cashmere fiber performance, including binding, cell, cellular protein modification process, and Wnt, Notch, and MAPK signaling pathways. The miRNA-mRNA interaction network found that 14 miRNAs selected may contribute to cashmere fiber traits regulation by targeting functional genes associated with hair follicle activities. The results have reinforced others leading to a solid foundation for further investigation of the influences of individual miRNAs on cashmere fiber traits in cashmere goats.

MicroRNA-199a-3p regulates proliferation and milk fat synthesis of ovine mammary epithelial cells by targeting VLDLR.

In our previous study, microRNA (miR)-199a-3p was found to be the most upregulated miRNA in mammary gland tissue during the non-lactation period compared with the peak-lactation period. However, there have been no reports describing the function of miR-199a-3p in ovine mammary epithelial cells (OMECs) and the biological mechanisms by which the miRNA affects cell proliferation and milk fat synthesis in sheep. In this study, the effect of miR-199a-3p on viability, proliferation, and milk fat synthesis of OMECs was investigated, and the target relationship of the miRNA with very low-density lipoprotein receptor (VLDLR) was also verified. Transfection with a miR-199a-3p mimic increased the viability of OMECs and the number of Edu-labeled positive OMECs. In contrast, a miR-199-3p inhibitor had the opposite effect with the miR-199a-3p mimic. The expression levels of three marker genes were also regulated by both the miR-199a-3p mimic and miR-199-3p inhibitor in OMECs. Together, these results suggest that miR-199a-3p promotes the viability and proliferation of OMECs. A dual luciferase assay confirmed that miR-199a-3p can target VLDLR by binding to the 3'-untranslated regions (3'UTR) of the gene. Further studies found a negative correlation in the expression of miR-199a-3p with VLDLR. The miR-199a-3p mimic decreased the content of triglycerides, as well as the expression levels of six milk fat synthesis marker genes in OMECs, namely, lipoprotein lipase gene (LPL), acetyl-CoA carboxylase alpha gene (ACACA), fatty acid binding protein 3 gene (FABP3), CD36, stearoyl-CoA desaturase gene (SCD), and fatty acid synthase gene (FASN). The inhibition of miR-199a-3p increased the level of triglycerides and the expression of LPL, ACACA, FABP3, SCD, and FASN in OMECs. These findings suggest that miR-199a-3p inhibited milk fat synthesis of OMECs. This is the first study to reveal the molecular mechanisms by which miR-199a-3p regulates the proliferation and milk fat synthesis of OMECs in sheep.

Tissue-Specific Expression of Circ_015343 and Its Inhibitory Effect on Mammary Epithelial Cells in Sheep.

Circular RNAs (circRNAs) are a kind of non-coding RNA that have an important molecular function in mammary gland development and lactation of mammals. In our previous study, circ_015343 was found to be highly expressed in the ovine mammary gland tissue at the peak-lactation period by using RNA sequencing (RNA-seq). In the present study, the authenticity of circ_015343 was confirmed by using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and Sanger sequencing. The circ_015343 was derived from the complete 10 exons of aminoadipic semialdehyde synthase (AASS), ranging from exon 2 to exon 11 and mainly located in cytoplasm of ovine mammary epithelial cells. The circRNA was found to be expressed in eight ovine tissues, with the highest expression level in the mammary gland and the least expression in Longissimus dorsi muscle. The circ_015343 had a lower level of expression in a sheep breed with higher milk yield and milk fat content. The disturbed circ_015343 increased the viability and proliferation of the ovine mammary epithelial cells. The inhibition of circ_015343 also increased the expression levels of three milk fat synthesis marker genes: acetyl-coenzyme A carboxylase alpha (ACACA), fatty acid-binding protein 4 (FABP4), and sterol regulatory element-binding protein 1 (SREBP1), as well as three proliferation-related genes: cyclin dependent kinase 2 (CDK2), cyclin dependent kinase 4 (CDK4) and proliferating cell nuclear antigen (PCNA), but decreased the expression level of its parent gene AASS. A circRNA-miRNA-mRNA interaction network showed that circ_015343 would bind some microRNAs (miRNAs) to regulate the expression of functional genes related to the development of mammary gland and lactation. This study contributes to a better understanding of the roles of circ_015343 in the mammary gland of sheep.

MicroRNA-148a Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting PTEN.

MicroRNAs (miRNAs) have been found to be involved in lipid deposition and metabolism. However, there have been no reports on the roles of miR-148a in the proliferation and adipogenesis of preadipocytes in sheep. In this study, the expression of miR-148a was profiled in the eight tissues of Tibetan ewes and differentiated preadipocytes, and the role of miR-148a in differentiation and proliferation of ovine preadipocytes was investigated using Oil Red O staining, CCK-8, EdU staining, cell cycle detection, and RT-qPCR. The effect of PTEN on the differentiation of ovine preadipocytes was also investigated. The miR-148a was widely expressed in the eight tissues investigated and had significantly increased expression in liver, spleen and subcutaneous adipose tissues, and the heart. The expression of miR-148a continued to increase with the differentiation of ovine preadipocytes. The over-expression of miR-148a significantly promoted differentiation but inhibited the proliferation of ovine preadipocytes. The inhibition of miR-148a had the opposite effect on the differentiation and proliferation of ovine preadipocytes with over-expressed miR-148a. The results from the dual luciferase reporter assays showed that miR-148a mimic significantly decreased the luciferase activity of PTEN-3'UTR dual luciferase reporter vector, suggesting that PTEN is a target gene of miR-148a. In over-expressed-PTEN preadipocytes, the number of lipid droplets remarkably decreased, and the expression levels of adipogenesis marker genes PPARγ, FASN, FATP4, GLUT4, C/EBPß and LPL were also significantly down-regulated. These results suggest that miR-148a accelerated the adipogenic differentiation of ovine preadipocytes by inhibiting PTEN expression, and also inhibited the proliferation of ovine preadipocytes.

Identification and characterization of circular RNAs in mammary gland tissue from sheep at peak lactation and during the nonlactating period.

Circular RNAs are a class of noncoding RNA with a widespread occurrence in eukaryote tissues, and with some having been demonstrated to have clear biological function. In sheep, little is known about the role of circular RNAs in mammary gland tissue, and therefore an RNA sequencing approach was used to compare mammary gland tissue expression of circular RNAs in 9 Small Tail Han sheep at peak lactation, and subsequently when they were not lactating. These 9 sheep had their RNA pooled for analysis into 3 libraries from peak lactation and 3 from the nonlactating period. A total of 3,278 and 1,756 circular RNAs were identified in the peak lactation and nonlactating mammary gland tissues, respectively, and the expression and identity of 9 of them was confirmed using reverse transcriptase-polymerase chain reaction analysis and DNA sequencing. The type, chromosomal location and length of the circular RNAs identified were ascertained. Forty upregulated and one downregulated circular RNAs were characterized in the mammary gland tissue at peak lactation compared with the nonlactating mammary gland tissue. Gene ontology enrichment analysis revealed that the parental genes of these differentially expressed circular RNAs were related to molecular function, binding, protein binding, ATP binding, and ion binding. Five differentially expression circular RNAs were selected for further analysis to predict their target microRNAs, and some microRNAs reportedly associated with the development of the mammary gland were found in the constructed circular RNA-microRNA network. This study reveals the expression profiles and characterization of circular RNAs at 2 key stages of mammary gland activity, thereby providing an improved understanding of the roles of circular RNAs in the mammary gland of sheep.

RNA-Seq Reveals the Expression Profiles of Long Non-Coding RNAs in Lactating Mammary Gland from Two Sheep Breeds with Divergent Milk Phenotype.

Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA with >200 nucleotides in length. Some lncRNAs have been proven to have clear regulatory functions in many biological processes of mammals. However, there have been no reports on the roles of lncRNAs in ovine mammary gland tissues. In the study, the expression profiles of lncRNAs were studied using RNA-Seq in mammary gland tissues from lactating Small-Tailed Han (STH) ewes and Gansu Alpine Merino (GAM) ewes with different milk yield and ingredients. A total of 1894 lncRNAs were found to be expressed. Compared with the GAM ewes, the expression levels of 31 lncRNAs were significantly up-regulated in the mammary gland tissues of STH ewes, while 37 lncRNAs were remarkably down-regulated. Gene Ontogeny (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the target genes of differentially expressed lncRNAs were enriched in the development and proliferation of mammary epithelial cells, morphogenesis of mammary gland, ErbB signaling pathway, and Wnt signaling pathway. Some miRNA sponges of differentially expressed lncRNAs, reported to be associated with lactation and mammary gland morphogenesis, were found in a lncRNA-miRNA network. This study reveals comprehensive lncRNAs expression profiles in ovine mammary gland tissues, thereby providing a further understanding of the functions of lncRNAs in the lactation and mammary gland development of sheep.

Identification of the Ovine Keratin-Associated Protein 2-1 Gene and Its Sequence Variation in Four Chinese Sheep Breeds.

Keratin-associated proteins are important components of wool fibers. The gene encoding the high-sulfur keratin-associated protein 2-1 has been described in humans, but it has not been described in sheep. A basic local alignment search tool nucleotide search of the Ovine Genome Assembly version 4.0 using a human keratin-associated protein 2-1 gene sequence revealed a 399-base pair open reading frame, which was clustered among nine previously identified keratin-associated protein genes on chromosome 11. Polymerase chain reaction-single strand conformation polymorphism analysis revealed four different banding patterns, with these representing four different sequences (A-D) in Chinese sheep breeds. These sequences had the highest similarity to human keratin-associated protein 2-1 gene, suggesting that they represent variants of ovine keratin-associated protein 2-1 gene. Nine single nucleotide variations were detected in the gene, including one non-synonymous nucleotide substitution. Differences in variant frequencies between fine-wool sheep breeds and coarse-wool sheep breeds were detected. The gene was found to be expressed in various tissues, with the highest expression level in skin, and moderate expression levels in heart and lung tissue. These results reveal that the ovine keratin-associated protein 2-1 gene is variable and suggest the gene might affect variation in mean fiber diameter.

Comparative Transcriptome Profile Analysis of Longissimus dorsi Muscle Tissues From Two Goat Breeds With Different Meat Production Performance Using RNA-Seq.

Carcass weight, meat quality and muscle components are important traits economically and they underpin most of the commercial return to goat producers. In this study, the Longissimus dorsi muscle tissues were collected from five Liaoning cashmere (LC) goats and five Ziwuling black (ZB) goats with phenotypic difference in carcass weight, some meat quality traits and muscle components. The histological quantitative of collagen fibers and the transcriptome profiles in the Longissimus dorsi muscle tissues were investigated using Masson-trichrome staining and RNA-Seq, respectively. The percentage of total collagen fibers in the Longissimus dorsi muscle tissues from ZB goats was less than those from LC goats, suggesting that these ZB goats had more tender meat. An average of 15,919 and 15,582 genes were found to be expressed in Longissimus dorsi muscle tissues from LC and ZB goats, respectively. Compared to LC goats, the expression levels of 78 genes were up-regulated in ZB goats, while 133 genes were down-regulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in GO terms related to the muscle growth and development and the deposition of intramuscular fat and lipid metabolism, hippo signaling pathway and Jak-STAT signaling pathway. The results provide an improved understanding of the genetic mechanisms regulating meat production performance in goats, and will help us improve the accuracy of selection for meat traits in goats using marker-assisted selection based on these differentially expressed genes obtained.